Vasoactive lntestinal Peptide Release From Microparticles

ABSTRACT

Controlled release of VIP from PLGA microparticles was accomplished and varied through use of different polymer molecular sizes, addition of solutes to the inner aqueous phase, and use of our computer model. Released VIP from microparticles appeared to be bioactive and caused DCs to produce more CCL22 than DCs treated with blank particles at 7 and 24 hours. Additionally, DCs treated with VIP microparticle releasates recruited higher percentages of FoxP3+ T-cells in in vitro chemotaxis studies. Testing in a mouse model in vivo indicated that VIP microparticles have significant therapeutic potential to treat periodontal disease by reducing the bone loss in infected mice relative to the blank group.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under grant numbersRR024154 and AI067541 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention is related to the treatment of immunologicaldisorders, immunological diseases and/or transplantation rejectionreactions. For example, certain embodiments of the invention result inthe regulation of blood cells including, but not limited, to naïve bloodcells and/or white blood cells (e.g., T cells and/or B cells) controlledby vasoactive intestinal peptide (VIP). In particular, the inventioncontemplates methods of creating VIP-induced regulatory T cells bycontrolled VIP release from custom designed microparticles.

BACKGROUND

Periodontal disease, also known as gingivitis or gum disease, involvesthe inflammation and destruction of the tissues that support the teeth.It is the leading cause of tooth loss and affects over 78 millionAmericans. Current treatments focus on removing bacteria associated withthe onset of the disease through clinical procedures known as scalingand root planing. However, recent studies have demonstrated that whilethe bacteria initiates periodontal disease, an imbalance of the immunesystem propagates the damage, specifically through an absence of asubset of lymphocytes called regulatory T cells (Tregs). Cardoso et al.,(2004) “Characterization of CD4+CD25+ natural regulatory T cells in theinflammatory infiltrate of human chronic periodontitis” Journal ofLeukocyte Biology 84(1):311-318. Garlet et al. found that CD4+CD25+ andCD4+FoxP3+ regulatory T cells attenuate the severity of periodontitis inmice. Garlet also found that an inhibition of these cells wascharacterized by an increase in alveolar bone loss and diseaseprogression, suggesting that restoration of regulatory T cells in theperiodontium may alleviate symptoms and restore immunological balance.Garlet et al., (2010) “Regulatory T cells attenuate experimentalperiodontitis progression in mice” Journal of Clinical Periodontology,37:591-600.

The science of transplantation, now half of a century old, hasdramatically increased and improved the life of many individuals,including many children, with end stage diseases. Recent advancements inimmunosuppressive agents have substantially decreased rejection ofallografts over the past decade and a half in the United States.Hariharan et al., N Engl J Med 342, 605-612 (2000); and First, M. R.,Transplant Proc 34:1369-1371 (2002). However, to avoid both episodes ofacute rejection and the initiation of chronic rejection followingtransplantation, immunosuppressive drugs must be administered over theentire life of the organ recipient.

Consequences of long-term immunosuppressive drug administration areprofound, including undesirable side effects, increasing the risk ofinfection, autoimmunity, heart disease, diabetes, and cancer. Thechronic administration of these immunosuppressive drugs (especially whengiven systemically) lead to toxicity and significant side effects,thereby leaving the patient vulnerable to a variety of diseases andsystemic organ failure. The most desirable alternative to this extendedstate of vulnerability would be to render the patient's immune system toeffectively suppress immune activation without systemicimmunosuppression. In this case, no further immunosuppressant drugtreatment would be necessary. Furthermore, the recipient's immune systemwould otherwise function normally, being capable of combating pathogensand malignant tumor cells.

What is needed in the art are compositions and methods to specificallyinduced regulatory T cells to treat tissues affected by immunologicaldiseases including, but not limited to, those related to inflammatoryconditions and/or tissue transplant rejection.

SUMMARY OF THE INVENTION

The present invention is related to the treatment of immunologicaldisorders, immunological diseases and/or transplantation rejectionreactions. For example, certain embodiments of the invention result inthe regulation of blood cells including but not limited to naïve bloodcells and/or white blood cells (e.g., T cells and/or B cells) controlledby vasoactive intestinal peptide (VIP). In particular, the inventioncontemplates methods of creating VIP-induced regulatory T cells bycontrolled VIP release from custom designed microparticles.

In one embodiment, the present invention contemplates a microparticlecomprising at least three polymers, wherein each of said three polymershas a different molecular weight. In one embodiment, said at least threepolymers have a molecular weight ranging between approximately 4-70 kDa50:50 PLGA. In one embodiment, one of said at least three polymersranges between approximately 7-17 kDa 50:50 PLGA (e.g., for example,12.6 kDa RG502H). In one embodiment, one of said at least three polymersranges between approximately 54-69 kDa 50:50 PLGA (e.g., for example,100 kDa RG505).

In one embodiment, the present invention contemplates a method,comprising: a) providing; i) a patient comprising a target tissue and ablood cell population, wherein said target tissue exhibits at least onesymptom of a disease; and ii) a microparticle population comprising avasoactive intestinal peptide; b) administering said microparticlepopulation to said patient under conditions such that said blood cellpopulation is induced; and c) contacting said induced blood cellpopulation with said target tissue such that said at least one symptomof said disease is reduced. In one embodiment, the disease comprises animmunological disease. In one embodiment, the blood cell is a whiteblood cell. In one embodiment, the white blood cell is a T cell. In oneembodiment, the T cell is a regulatory T cell. In one embodiment, thewhite blood cell is a B cell. In one embodiment, the microparticlepopulation comprises a plurality of polymers in a ratio predicted by amathematical algorithm. In one embodiment, the plurality of polymerscomprise a 4.2 kDa polymer, a 12.6 kDa polymer and a 55 kDa polymer. Inone embodiment, the plurality of polymers comprise a 4.2 kDa polymer, a12.6 kDa polymer and a 100 kDa polymer. In one embodiment, said ratio is1:3:5.4. In one embodiment, said ratio is 1:1:1.

In one embodiment, the present invention contemplates, a microparticlecomprising a 4.2 kDa polymer, a 12.6 kDa polymer and a 100 kDa polymerand a vasoactive intestinal peptide. In one embodiment, said 4.2 kDapolymer comprises 10.6% of said microparticle. In one embodiment, said12.6 kDa polymer comprises 31.9% of said microparticle. In oneembodiment, said 100 kDa polymer comprises 57.5% of said microparticle.In one embodiment, the microparticle further comprises an aqueous innerphase. In one embodiment, the aqueous inner phase comprises polyethyleneglycol. In one embodiment, the aqueous inner phase comprises sodiumchloride. In one embodiment, said 4.2 kDa polymer includes, but is notlimited to, a polylactide-co-glycolide (PLGA) polymer, an RG502H polymerand an RG505 polymer. In one embodiment, said 12.6 kDa polymer includes,but is not limited to, a polylactide-co-glycolide (PLGA) polymer, anRG502H polymer and an RG505 polymer. In one embodiment, said 100 kDapolymer includes, but is not limited to, a polylactide-co-glycolide(PLGA) polymer, an RG502H polymer and an RG505 polymer.

In one embodiment, the present invention contemplates a microparticlecomprising a 4.2 kDa polymer, a 12.6 kDa polymer and a 55 kDa polymerand vasoactive intestinal peptide. In one embodiment, said 4.2 kDapolymer comprises 33.3% of said microparticle. In one embodiment, said12.6 kDa polymer comprises 33.3% of said microparticle. In oneembodiment, said 55 kDa polymer comprises 33.3% of said microparticle.In one embodiment, the 12.6 kDa polymer is an RG502H polymer. In oneembodiment, the 55 kDA polymer is an RG505 polymer. In one embodiment,the microparticle further comprises an aqueous inner phase. In oneembodiment, the aqueous inner phase comprises polyethylene glycol. Inone embodiment, the aqueous inner phase comprises sodium chloride.

In one embodiment, the present invention contemplates a kit comprising:a) a first container comprising a composition comprising a microparticlepopulation encapsulating a vasoactive intestinal peptide; b) a secondcontainer comprising a pharmaceutically acceptable vehicle foradministration of said composition; and c) instructions foradministering said composition to a patient comprising a target tissueexhibiting at least one symptom of a disease. In one embodiment, themicroparticle population comprises a ratio of polymers predicted by amathematical algorithm. In one embodiment, the ratio of polymerscomprise a 4.2 kDa polymer, a 12.6 kDa polymer and a 55 kDa polymer. Inone embodiment, the ratio of polymers comprise a 4.2 kDa polymer, a 12.6kDa polymer and a 100 kDa polymer. In one embodiment, said ratio is1:3:5.4. In one embodiment, said ratio is 1:1:1. In one embodiment, thepolymer includes, but is not limited to, a polylactide-co-glycolide(PLGA) polymer, an RG502H polymer and/or an RG505 polymer. In oneembodiment, the microparticle further comprises an aqueous inner phase.In one embodiment, the aqueous inner phase comprises polyethyleneglycol. In one embodiment, the aqueous inner phase comprises sodiumchloride.

The present invention is related to the field of inducing immunologicaltolerance by specifically manipulating immune-related cells. Suchimmunological tolerance may be induced by providing biomimeticartificial cells that bypass native immunological cells. For example,biomimetic artificial cell compositions are contemplated which comprisesoluble factors that activate specific immune-related blood cells,including, but not limited to, macrophages and/or monocytes. Suchfactors may comprise chemoattractant factors. These biomimeticartificial particles may further present a specific biomimetic surfacepattern that results in a targeted cell response such that the particlesrepresent artificial presenting cells.

In one embodiment, the present invention contemplates an artificialparticle comprising a plurality of soluble and surface bound regulatoryT-cell factors. In one embodiment, the artificial particle comprises anartificial presenting cell. In one embodiment, the factors compriseregulatory T cell stimulatory factors. In one embodiment, the factorscomprise regulatory T cell inducing factors. In one embodiment, thefactors comprise regulatory T cell chemoattractant factors. In oneembodiment, the soluble factors undergo controlled released. In oneembodiment, the controlled release is a short term release. In oneembodiment, the short term release is between 30 minutes-1 hour. In oneembodiment, the short term release is between 1 hour-3 hours. In oneembodiment, the short term release is between 3 hours-10 hours. In oneembodiment, the short term release is between 10 hours-24 hours. In oneembodiment, the controlled release is a long term release. In oneembodiment, the long term release is between 24 hours-36 hours. In oneembodiment, the long term release is between 3 days-7 days. In oneembodiment, the long term release is between 7 days-1 month. In oneembodiment, the long term release is between 1 month-6 months. In oneembodiment, the long term release is between 6 months-1 year. In oneembodiment, the long term release is at least one year. In oneembodiment, the surface bound factors comprises a biomimetic surfacepattern. In one embodiment, the biomimetic surface pattern comprises animmunological synapse. In one embodiment, the artificial presenting celltake a shape selected from the group consisting of a sphere, a square, arectangle, a triangle, a trapezoid, a hexagon, an octagon, or atetrahedron.

In one embodiment, the present invention contemplates an artificialparticle comprising at least one soluble T_(reg) cell factor and atleast one surface bound T_(reg) cell factor, wherein said surface boundfactor is displayed in a non-random pattern. In one embodiment, theartificial particle comprises an artificial presenting cell. In oneembodiment, the at least one soluble or surface bound Treg cell factorcomprises a regulatory T cell stimulatory factor. In one embodiment, theat least one soluble or surface bound Treg cell factor comprises aregulatory T cell inducing factor. In one embodiment, the at least onesoluble or surface bound Treg cell factor comprises a regulatory T cellchemoattractant factor. In one embodiment, the artificial presentingcell further comprises a functionalized polymer, wherein said polymerencapsulates the soluble factor. In one embodiment, the artificialpresenting cell further comprises compartments, wherein saidcompartments comprise the at least one soluble factor. In oneembodiment, the artificial presenting cell is biodegradable such thatthe soluble factor undergoes controlled release. In one embodiment, thesoluble factor comprises CCL22. In one embodiment, the soluble factorcomprises IL2. In one embodiment, the soluble factor comprises TGF-β. Inone embodiment, the polymer comprises poly-lactic acid-co-glycolic acid(PLGA). In one embodiment, the functionalized polymer non-covalentlybonds the soluble factor. In one embodiment, the at least one surfacebound T_(reg) stimulating factor forms a biomimetic surface pattern. Inone embodiment, the biomimetic surface pattern comprises animmunological synapse. In one embodiment, the synapse comprises a cSMACregion, displayed on an apical portion of the artificial presentingcell. In one embodiment, the synapse comprises a pSMAC region displayedon a tropical region of the artificial presenting cell. In oneembodiment, the synapse comprises a dSMAC region displayed on anequatorial portion of the artificial presenting cell. In one embodiment,the cSMAC region comprises CD3 antibodies and/or CD28 antibodies. In oneembodiment, the pSMAC region comprises at least one adhesion molecule.In one embodiment, the dSMAC region comprises a CD45 monoclonalantibody. In one embodiment, the functionalized polymer covalently bondsthe surface bound regulatory T cell stimulatory factor.

In one embodiment, the present invention contemplates a method,comprising: a) providing, i) an emulsified microparticle comprising atleast one soluble T_(reg) cell factor and a functionalized polymer; andii) at least one surface bound T_(reg) cell factor, wherein the surfacebound factor is capable of attaching to the polymer; b) attaching afirst surface bound factor to said polymer, wherein an apical region iscreated; c) attaching a second surface bound factor to said polymer,wherein a tropical region is created; and d) attaching a third surfacebound factor to said polymer, wherein an equatorial region is created.In one embodiment, the microparticle comprises an artificial presentingcell. In one embodiment, the at least one soluble or surface bound Tregcell factor comprises a regulatory T cell stimulatory factor. In oneembodiment, the at least one soluble or surface bound Treg cell factorcomprises a regulatory T cell inducing factor. In one embodiment, the atleast one soluble or surface bound Treg cell factor comprises aregulatory T cell chemoattractant factor. In one embodiment, themicroparticle is biodegradable such that the soluble factor undergoescontrolled release. In one embodiment, the soluble factor comprisesCCL22. In one embodiment, the soluble factor comprises IL2. In oneembodiment, the soluble factor comprises TGF-13. In one embodiment, theapical, tropical, and equatorial regions comprise a biomimetic pattern.In one embodiment, the biomimetic pattern comprises an immunologicalsynapse. In one embodiment, the first surface bound factor is selectedfrom the group consisting of a CD3 antibody and a CD28 antibody. In oneembodiment, the second surface bound factor comprises an adhesionprotein. In one embodiment, the third surface bound factor comprises aCD45 antibody. Alternatively, the microparticle may further comprise aCD3 ligand (i.e., MHC) and/or a CD28 ligand.

In one embodiment, the present invention contemplates a method,comprising: a) providing, i) a polymer capable of undergoingemulsification, ii) at least one soluble T_(reg) cell factor, and iii)at least one surface bound T_(reg) cell factor; and b) emulsifying thepolymer and the soluble factor such that a biodegradable microparticleis created, such that the soluble factor undergoes controlled release.In one embodiment, the microparticle comprises an artificial presentingcell. In one embodiment, the at least one soluble or surface bound Tregcell factor comprises a regulatory T cell stimulatory factor. In oneembodiment, the at least one soluble or surface bound Treg cell factorcomprises a regulatory T cell inducing factor. In one embodiment, the atleast one soluble or surface bound Treg cell factor comprises aregulatory T cell chemoattractant factor. In one embodiment, theemulsifying may be selected from the group including, but not limitedto, precipitation, emulsions, melt casting, spray drying,crystallization, shearing, or milling. In one embodiment, themicroparticle is porous. In one embodiment, the microparticle comprisescompartment. In one embodiment, the method further comprises step (c)immobilizing a first surface bound stimulatory factor in an apicalregion of the microparticle. In one embodiment, the method furthercomprises step (d) immobilizing a second surface bound stimulatoryfactor in a tropical region of the microparticle. In one embodiment, themethod further comprises step (e) immobilizing a third surfacestimulatory factor in an equatorial region of the microparticle. In oneembodiment, the polymer comprises poly-lactic acid-co-glycolic acid(PLGA). In one embodiment, the soluble stimulatory factor is selectedfrom the group consisting of CCL22, IL2, and TGF-β. In one embodiment,the first surface bound factor is selected from the group consisting ofCD3 antibody and CD28 antibody. In one embodiment, the second surfacebound factor comprises an adhesion molecule. In one embodiment, thethird surface bound factor comprises a CD45 antibody. In one embodiment,the at least one surface bound signaling factor is randomly displayed onthe microparticle.

In one embodiment, the present invention contemplates a method,comprising: a) providing, i) a patient having received a tissuetransplant, wherein said tissue is capable of immunolgical tolerance;ii) an artificial antigen presenting cell comprising at least onesoluble T_(reg) cell factor and at least one surface bound T_(reg) cellfactor; and b) administering the artificial antigen presenting cell tothe patient under conditions such that an immunological tolerant stateis induced in the tissue. In one embodiment, the at least one soluble orsurface bound Treg cell factor comprises a regulatory T cell stimulatoryfactor. In one embodiment, the at least one soluble or surface boundTreg cell factor comprises a regulatory T cell inducing factor. In oneembodiment, the at least one soluble or surface bound Treg cell factorcomprises a regulatory T cell chemoattractant factor. In one embodiment,the artificial antigen presenting cell is porous. In one embodiment, theimmunosuppression is not antigen-specific. In one embodiment, theartificial antigen presenting cell comprises compartments. In oneembodiment, the compartments comprise the soluble factor. In oneembodiment, the artificial antigen presenting cell is biodegradable,wherein the soluble factor undergoes controlled release. In oneembodiment, the controlled release is a short term release. In oneembodiment, the short term release is between 30 minutes-1 hour. In oneembodiment, the short term release is between 1 hour-3 hours. In oneembodiment, the short term release is between 3 hours-10 hours. In oneembodiment, the short term release is between 10 hours-24 hours. In oneembodiment, the controlled release is a long term release. In oneembodiment, the long term release is between 24 hours-36 hours. In oneembodiment, the long term release is between 3 days-7 days. In oneembodiment, the long term release is between 7 days-1 month. In oneembodiment, the long term release is between 1 month-6 months. In oneembodiment, the long term release is between 6 months-1 year. In oneembodiment, the long term release is at least one year. In oneembodiment, the method further comprises step (c) inducing a tissuetolerance by the immunosuppressive state. In one embodiment, the tissuetolerance is permanent. In one embodiment, the soluble stimulatoryfactor is selected from the group consisting of CCL22, IL2, and TGF-β.In one embodiment, the surface bound factors are selected from the groupconsisting of CD3 antibody, CD28 antibody, an adhesion molecule, and aCD45 antibody. Alternatively, the microparticle may further comprise aCD3 ligand (i.e., MHC) and/or a CD28 ligand.

In one embodiment, the present invention contemplates a kit comprising acontainer comprising at least one artificial particle suspended in apharmaceutically acceptable vehicle. In one embodiment, the artificialparticle comprises an artificial presenting cell. In one embodiment, thekit further comprises instructions for using the artificial particle toinduce immunosuppression in a patient. In one embodiment, the artificialparticle comprises at least one encapsulated soluble T_(reg) factor. Inone embodiment, the at least one soluble or surface bound Treg cellfactor comprises a regulatory T cell stimulatory factor. In oneembodiment, the at least one soluble or surface bound Treg cell factorcomprises a regulatory T cell inducing factor. In one embodiment, the atleast one soluble or surface bound Treg cell factor comprises aregulatory T cell chemoattractant factor. In one embodiment, the solublefactor is selected from the group consisting of CCL22, IL2 or TGF-β.

In one embodiment, the present invention contemplates an artificialosteoblast cell comprising a plurality of soluble and surface boundosteoclast signaling factors. In one embodiment, the soluble signalingfactors are controlled released. In one embodiment, the solublesignaling factor comprises MCSF. In one embodiment, the surface boundsignaling factor comprises RANK-L, OSCAR-L, and/or ODF. In oneembodiment, the controlled release is a short term release. In oneembodiment, the short term release is between 30 minutes-1 hour. In oneembodiment, the short term release is between 1 hour-3 hours. In oneembodiment, the short term release is between 3 hours-10 hours. In oneembodiment, the short term release is between 10 hours-24 hours. In oneembodiment, the controlled release is a long term release. In oneembodiment, the long term release is between 24 hours-36 hours. In oneembodiment, the long term release is between 3 days-7 days. In oneembodiment, the long term release is between 7 days-1 month. In oneembodiment, the long term release is between 1 month-6 months. In oneembodiment, the long term release is between 6 months-1 year. In oneembodiment, the long term release is at least one year. In oneembodiment, the surface bound signaling factors comprises a biomimeticsurface pattern. In one embodiment, the biomimetic surface patterncomprises an osteological synapse. In one embodiment, the artificialosteoblast cell take a shape selected from the group consisting of asphere, a square, a rectangle, a triangle, a trapezoid, a hexagon, anoctagon, or a tetrahedron.

In one embodiment, the present invention contemplates an artificialosteoblast cell comprising at least one soluble osteoclast cellsignaling factor and at least one surface bound osteoclast cellsignaling factor. In one embodiment, the artificial osteoblast cellfurther comprises a functionalized polymer, wherein said polymerencapsulates the soluble factor. In one embodiment, the artificialosteoblast cell further comprises compartments, wherein saidcompartments comprise the soluble signaling factor. In one embodiment,the artificial osteoblast cell is biodegradable such that the solublefactor undergoes controlled release. In one embodiment, the solublefactor comprises MCSF. In one embodiment, the polymer comprisespoly-lactic acid-co-glycolic acid (PLGA). In one embodiment, thefunctionalized polymer non-covalently bonds the soluble factor. In oneembodiment, the at least one surface bound osteoclast signaling factorforms a biomimetic surface pattern. In one embodiment, the biomimeticsurface pattern comprises an osteological synapse. In one embodiment,the surface bound osteoclast signaling factor comprises RANK-L, OSCAR-L,and/or ODF. In one embodiment, the functionalized polymer covalentlybonds the surface bound osteoclast cell stimulatory factor.

In one embodiment, the present invention contemplates a method,comprising: a) providing, i) an emulsified microparticle comprising atleast one soluble osteoclast cell signaling factor and a functionalizedpolymer; and ii) at least one surface bound osteoclast cell signalingfactor, wherein the surface bound signaling factor is capable ofattaching (i.e., for example, by conjugating and/or adsorbing) to thepolymer; b) attaching a first surface bound factor to said polymer,wherein a first region is created; c) attaching a second surface boundfactor to said polymer, wherein a second region is created; and d)attaching a third surface bound factor to said polymer, wherein a thirdregion is created. In one embodiment, the microparticle is biodegradablesuch that the soluble factor undergoes controlled release. In oneembodiment, the at least one soluble signaling factor comprises MSCF. Inone embodiment, the first, second, and third regions comprise abiomimetic pattern. In one embodiment, the biomimetic pattern comprisesan osteological synapse. In one embodiment, the first surface boundsignaling factor comprises RANK-L, OSCAR-L, and/or ODF.

In one embodiment, the present invention contemplates a method,comprising: a) providing, i) a polymer capable of undergoingemulsification, ii) at least one soluble osteoclast cell signalingfactor, and iii) at least one surface bound osteoclast cell signalingfactor; and b) emulsifying the polymer and the soluble signaling factorsuch that a biodegradable microparticle is created, such that thesoluble factor undergoes controlled release. In one embodiment, themicroparticle is porous. In one embodiment, the microparticle comprisescompartments. In one embodiment, the emulsifying may be selected fromthe group including, but not limited to, precipitation, emulsions, meltcasting, spray drying, crystallization, shearing, or milling. In oneembodiment, the method further comprises step (c) immobilizing a firstsurface bound signaling factor in an apical region of the microparticle.In one embodiment, the method further comprises step (d) immobilizing asecond surface bound signaling factor in a tropical region of themicroparticle. In one embodiment, the method further comprises step (e)immobilizing a third surface signaling factor in an equatorial region ofthe microparticle. In one embodiment, the polymer comprises poly-lacticacid-co-glycolic acid (PLGA). In one embodiment, the soluble signalingfactor comprises MCSF. In one embodiment, the at least one surface boundsignaling factor comprises RANK-L, OSCAR-L, and/or ODF. In oneembodiment, the at least one surface bound signaling factor is randomlydisplayed on the microparticle.

In one embodiment, the present invention contemplates a method,comprising: a) providing, i) a patient, wherein said patient is in needof bone healing; ii) an artificial osteoblast cell comprising at leastone soluble osteoclast cell signaling factor and at least one surfacebound osteoclast cell signaling factor; and b) administering theartificial osteoblast cell to the patient under conditions such that thebone healing is induced in the patient. In one embodiment, the patienthas under gone bone surgery. In one embodiment, the patient comprises abone injury. In one embodiment, the patient comprises a bone disease. Inone embodiment, the artificial osteoblast cell is porous. In oneembodiment, the artificial osteoblast cell comprises compartments. Inone embodiment, the compartments comprise the soluble signaling factor.In one embodiment, the artificial osteoblast cell is biodegradable,wherein the soluble factor undergoes controlled release. In oneembodiment, the controlled release is a short term release. In oneembodiment, the short term release is between 30 minutes-1 hour. In oneembodiment, the short term release is between 1 hour-3 hours. In oneembodiment, the short term release is between 3 hours-10 hours. In oneembodiment, the short term release is between 10 hours-24 hours. In oneembodiment, the controlled release is a long term release. In oneembodiment, the long term release is between 24 hours-36 hours. In oneembodiment, the long term release is between 3 days-7 days. In oneembodiment, the long term release is between 7 days-1 month. In oneembodiment, the long term release is between 1 month-6 months. In oneembodiment, the long term release is between 6 months-1 year. In oneembodiment, the long term release is at least one year. In oneembodiment, the bone surgery comprises bone replacement including, butnot limited to, hip replacement or knee replacement. In one embodiment,the bone surgery comprises bone reconstruction. In one embodiment, thebone surgery comprises bone fracture repair. In one embodiment, thesoluble signaling factor comprises MCSF. In one embodiment, the surfacebound signaling factor comprises RANK-L, OSCAR-L, and/or ODF.

In one embodiment, the present invention contemplates a kit comprising acontainer comprising at least one artificial osteoblast cell suspendedin a pharmaceutically acceptable vehicle. In one embodiment, the kitfurther comprises instructions for using the artificial osteoblast cellto induce bone healing in a patient. In one embodiment, the artificialosteoblast cell comprises at least one encapsulated soluble osteoclastsignaling factor. In one embodiment, the soluble signaling factorcomprises MCSF.

In one embodiment, the present invention contemplates an artificialosteoclast cell comprising a plurality of soluble and surface boundosteoblast signaling factors. In one embodiment, the soluble signalingfactors are controllably released. In one embodiment, the solublesignaling factor comprises MIM1. In one embodiment, the surface boundsignaling factor comprises EphrinB2 and/or TGF-β. In one embodiment, thecontrolled release is a short term release. In one embodiment, the shortterm release is between 30 minutes-1 hour. In one embodiment, the shortterm release is between 1 hour-3 hours. In one embodiment, the shortterm release is between 3 hours-10 hours. In one embodiment, the shortterm release is between 10 hours-24 hours. In one embodiment, thecontrolled release is a long term release. In one embodiment, the longterm release is between 24 hours-36 hours. In one embodiment, the longterm release is between 3 days-7 days. In one embodiment, the long termrelease is between 7 days-1 month. In one embodiment, the long termrelease is between 1 month-6 months. In one embodiment, the long termrelease is between 6 months-1 year. In one embodiment, the long termrelease is at least one year. In one embodiment, the surface boundsignaling factors comprises a biomimetic surface pattern. In oneembodiment, the biomimetic surface pattern comprises an osteologicalsynapse. In one embodiment, the artificial osteoblast cell take a shapeselected from the group consisting of a sphere, a square, a rectangle, atriangle, a trapezoid, a hexagon, an octagon, or a tetrahedron.

In one embodiment, the present invention contemplates an artificialosteoclast cell comprising at least one soluble osteoblast cellsignaling factor and at least one surface bound osteoblast cellsignaling factor. In one embodiment, the artificial osteoclast cellfurther comprises a functionalized polymer, wherein said polymerencapsulates the soluble factor. In one embodiment, the functionalizedpolymer comprises a surface polymer. In one embodiment, the artificialosteoclast cell further comprises compartments, wherein saidcompartments comprise the soluble signaling factor. In one embodiment,the artificial osteoclast cell is biodegradable such that the solublefactor undergoes controlled release. In one embodiment, the solublefactor comprises MIM1. In one embodiment, the polymer comprisespoly-lactic acid-co-glycolic acid (PLGA). In one embodiment, thefunctionalized polymer non-covalently bonds the soluble factor. In oneembodiment, the at least one surface bound osteoblast signaling factorforms a biomimetic surface pattern. In one embodiment, the biomimeticsurface pattern comprises an osteological synapse. In one embodiment,the surface bound osteoblast signaling factor comprises EphrinB2 and/orTGF-β. In one embodiment, the functionalized polymer covalently bondsthe surface bound osteoclast cell stimulatory factor.

In one embodiment, the present invention contemplates a method,comprising: a) providing, i) an emulsified microparticle comprising atleast one soluble osteoblast cell signaling factor and a functionalizedpolymer; and ii) at least one surface bound osteoblast cell signalingfactor, wherein the surface bound signaling factor is capable ofattaching to the polymer; b) attaching a first surface bound factor tosaid polymer, wherein an apical region is created; c) attaching a secondsurface bound factor to said polymer, wherein a tropical region iscreated; and d) attaching a third surface bound factor to said polymer,wherein an equatorial region is created. In one embodiment, themicroparticle is biodegradable such that the soluble factor undergoescontrolled release. In one embodiment, the at least one solublesignaling factor comprises MIM1. In one embodiment, the apical,tropical, and equatorial regions comprise a biomimetic pattern. In oneembodiment, the biomimetic pattern comprises an osteological synapse. Inone embodiment, the first surface bound signaling factor comprisesEphrinB2 and/or TGF-β.

In one embodiment, the present invention contemplates a method,comprising: a) providing, i) a polymer capable of undergoingemulsification, ii) at least one soluble osteoblast cell signalingfactor, and iii) at least one surface bound osteoblast cell signalingfactor; and b) emulsifying the polymer and the soluble signaling factorsuch that a porous biodegradable microparticle is created, such that thesoluble factor undergoes controlled release. In one embodiment, theemulsifying may be selected from the group including, but not limitedto, precipitation, emulsions, melt casting, spray drying,crystallization, shearing, or milling. In one embodiment, the methodfurther comprises step (c) immobilizing a first surface bound signalingfactor in an apical region of the microparticle. In one embodiment, themethod further comprises step (d) immobilizing a second surface boundsignaling factor in a tropical region of the microparticle. In oneembodiment, the method further comprises step (e) immobilizing a thirdsurface signaling factor in an equatorial region of the microparticle.In one embodiment, the polymer comprises poly-lactic acid-co-glycolicacid (PLGA). In one embodiment, the soluble signaling factor comprisesMINE. In one embodiment, the at least one surface bound signaling factorcomprises EphrinB2 and/or TGF-β.

In one embodiment, the present invention contemplates a method,comprising: a) providing, i) a patient having undergone bone surgery,wherein said patient is in need of bone healing; ii) a porous artificialosteoclast cell comprising at least one soluble osteoblast cellsignaling factor and at least one surface bound osteoblast cellsignaling factor; and b) administering the artificial osteoclast cell tothe patient under conditions such that a bone healing is induced in thepatient. In one embodiment, the porous artificial osteoclast cellcomprises compartments. In one embodiment, the compartments comprise thesoluble signaling factor. In one embodiment, the artificial osteoclastcell is biodegradable, wherein the soluble factor undergoes controlledrelease. In one embodiment, the controlled release is a short termrelease. In one embodiment, the short term release is between 30minutes-1 hour. In one embodiment, the short term release is between 1hour-3 hours. In one embodiment, the short tem release is between 3hours-10 hours. In one embodiment, the short term release is between 10hours-24 hours. In one embodiment, the controlled release is a long termrelease. In one embodiment, the long term release is between 24 hours-36hours. In one embodiment, the long term release is between 3 days-7days. In one embodiment, the long term release is between 7 days-1month. In one embodiment, the long term release is between 1 month-6months. In one embodiment, the long temp release is between 6 months-1year. In one embodiment, the long term release is at least one year. Inone embodiment, the bone surgery comprises bone replacement including,but not limited to, hip replacement or knee replacement. In oneembodiment, the bone surgery comprises bone reconstruction. In oneembodiment, the bone surgery comprises bone fracture repair. In oneembodiment, the soluble signaling factor comprises MIM1. In oneembodiment, the surface bound signaling factor comprises EphrinB2 and/orTGF-β.

In one embodiment, the present invention contemplates a kit comprising acontainer comprising at least one artificial osteoclast cell suspendedin a pharmaceutically acceptable vehicle. In one embodiment, the kitfurther comprises instructions for using the artificial osteoclast cellto induce bone healing in a patient. In one embodiment, the artificialosteoclast cell comprises at least one encapsulated soluble osteoblastsignaling factor. In one embodiment, the soluble signaling factorcomprises MIM1.

In one embodiment, the present invention contemplates a method,comprising: a) providing, i) a patient, wherein said patient comprisesat least one inflammatory symptom; ii) a microparticle comprising atleast one soluble Treg factor; and b) administering the microparticle tothe patient under conditions such that the at least one inflammatorysymptom is reduced. In one embodiment, the at least one soluble orsurface bound Treg factor comprises a regulatory T cell stimulatoryfactor. In one embodiment, the at least one soluble or surface boundTreg cell factor comprises a regulatory T cell inducing factor. In oneembodiment, the at least one soluble or surface bound Treg cell factorcomprises a regulatory T cell chemoattractant factor. In one embodiment,the patient has under gone periodontal surgery. In one embodiment, thepatient further comprises a tissue injury. In one embodiment, thepatient further comprises a periodontal disease. In one embodiment, thepatient comprises a cancer disease. In one embodiment, the microparticleis porous. In one embodiment, the microparticle is biodegradable,wherein the Treg chemoattractant factor undergoes controlled release. Inone embodiment, the controlled release is a short term release. In oneembodiment, the short term release is between 30 minutes-1 hour. In oneembodiment, the short term release is between 1 hour-3 hours. In oneembodiment, the short term release is between 3 hours-10 hours. In oneembodiment, the short term release is between 10 hours-24 hours. In oneembodiment, the controlled release is a long term release. In oneembodiment, the long term release is between 24 hours-36 hours. In oneembodiment, the long term release is between 3 days-7 days. In oneembodiment, the long term release is between 7 days-1 month. In oneembodiment, the long term release is between 1 month-6 months. In oneembodiment, the long term release is between 6 months-1 year. In oneembodiment, the long term release is at least one year. In oneembodiment, the Treg chemoattractant factor comprises CCL22 and/orvasoactive intestinal peptide.

DEFINITIONS

The term “target tissue” as used herein, refers to any bodily tissuethat may be affected by a medical condition and/or disorder (e.g., animmunological disease or a transplanted tissue/organ) to which apopulation of regulatory T cells may be directed to by induction with anindividual and/or a combination of T cell inducing factors released frommicroparticles.

The term “blood cell” as used herein, refers to any biological cell,either nucleated or enucleated, found circulating in the blood stream.

The term “white blood cell” as used herein, refers to any blood cellthat is colorless, lacks hemoglobin, contains a nucleus, and mayinclude, but is not limited to, lymphocytes, monocytes, neutrophils,eosinophils, and basophils—called also leukocytes, white bloodcorpuscles, white cells, and/or white corpuscles.

The term “T cell” as used herein, refers to any of several lymphocytetypes (e.g., helper T cell or regulatory T cell) that differentiate inthe thymus, possess highly specific cell-surface antigen receptors, thatmay control the initiation and/or suppression of cell-mediated andhumoral immunity (as by the regulation of T and B cell maturation andproliferation) and others that lyse antigen-bearing cells—also referredto as a T lymphocyte.

The term “B cell” as used herein, refers to any of the severallymphocytes that have antigen-binding antibody molecules on theirmembrane surface, that comprise the antibody-secreting plasma cells whenmature, and that in mammals differentiate in the bone marrow—alsoreferred to as a B lymphocyte.

The term “microparticle population” as used herein, refers to acollection of microparticles and/or microspheres having similarproperties and composition.

The term “polymer” as used herein, refers to any unit-based chain ofmolecules. For example, such molecules may include, but are not limitedto, gelatin, collagen, cellulose esters, dextran sulfate, pentosanpolysulfate, chitin, saccharides, albumin, synthetic polyvinylpyrrolidone, polyethylene oxide, polypropylene oxide, block polymers ofpolyethylene oxide and polypropylene oxide, polyethylene glycol,acrylates, acrylamides, methacrylates including, but not limited to,2-hydroxyethyl methacrylate, poly(ortho esters), cyanoacrylates,gelatin-resorcin-aldehyde type bioadhesives, polyacrylic acid andcopolymers and block copolymers thereof. Alternatively, polymers mayinclude, but are not limited to, poly(lactide-co-glycolide) polymer(e.g., PLGA), RG505, and/or RG502H. For example, one polymer may becomprised of at least three polymers ranging from 4-70 kDa 50:50 PLGA.Another polymer may be comprised of 7-17 kDa 50:50 PLGA (e.g., forexample, 12.6 kDa RG502H). Another polymer may be comprised of 54-69 kDa50:50 PLGA (e.g., for example, 100 kDa RG505).

The term “microparticle” as used herein, refers to any microscopiccarrier to which a compound or drug may be attached and/or encapsulated.Preferably, microparticles contemplated by this invention are capable offormulations having controlled release properties.

The term “attached” as used herein, refers to any interaction between amedium (or carrier) and a drug. Attachment may be reversible orirreversible. Such attachment includes, but is not limited to, covalentbonding, ionic bonding, Van der Waals forces or friction, and the like.A drug is attached to a medium (or carrier) if it is impregnated,incorporated, coated, in suspension with, in solution with, mixed with,etc.

The term “encapsulated” as used herein, refers to any composition wherea secondary compound (e.g., a drug, protein, peptide, nucleic acid etc.)is trapped within the composition and/or attached throughout thecomposition including the surface of the composition. Usually,encapsulation occurs when the secondary compound is present during theformation of the composition, as opposed to being contacted with thecomposition after the formation of the composition.

The term “PLGA” as used herein, refers to mixtures of polymers orcopolymers of lactic acid and glycolic acid. As used herein, lactidepolymers are chemically equivalent to lactic acid polymer and glycolidepolymers are chemically equivalent to glycolic acid polymers. In oneembodiment, PLGA contemplates an alternating mixture of lactide andglycolide polymers, and is referred to as a poly(lactide-co-glycolide)polymer.

The term “at risk for” as used herein, refers to a medical condition orset of medical conditions exhibited by a patient which may predisposethe patient to a particular disease or affliction. For example, theseconditions may result from influences that include, but are not limitedto, behavioral, emotional, chemical, biochemical, or environmentalinfluences.

The term “effective amount” as used herein, refers to a particularamount of a pharmaceutical composition comprising a therapeutic agentthat achieves a clinically beneficial result (i.e., for example, areduction of symptoms). Toxicity and therapeutic efficacy of suchcompositions can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index, and itcan be expressed as the ratio LD₅₀/ED₅₀. Compounds that exhibit largetherapeutic indices are preferred. The data obtained from these cellculture assays and additional animal studies can be used in formulatinga range of dosage for human use. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage varies within this rangedepending upon the dosage form employed, sensitivity of the patient, andthe route of administration.

The term “symptom” as used herein, refers to any subjective or objectiveevidence of disease or physical disturbance observed by the patient. Forexample, subjective evidence is usually based upon patientself-reporting and may include, but is not limited to, pain, headache,visual disturbances, nausea and/or vomiting. Alternatively, objectiveevidence is usually a result of medical testing including, but notlimited to, body temperature, complete blood count, lipid panels,thyroid panels, blood pressure, heart rate, electrocardiogram, tissueand/or body imaging scans.

The term “transplant rejection reaction” or “graft versus host disease”as used herein, refers to any activation of the immune system subsequentto the implantation of an exogenous tissue and/or organ into a patientthat may result in damage and/or destruction of the transplanted tissue.Generally, transplant rejections are believed to be an adaptive immuneresponse via cellular immunity (i.e., for example, mediated by killer Tcells inducing apoptosis of target cells) as well as humoral immunity(mediated by activated B cells secreting antibody molecules), though theaction is joined by components of innate immune response (phagocytes andsoluble immune proteins).

The term “disease” as used herein, refers to any impairment of thenormal state of the living animal or plant body or one of its parts thatinterrupts or modifies the performance of the vital functions. Typicallymanifested by distinguishing signs and symptoms, it is usually aresponse to: i) environmental factors (as malnutrition, industrialhazards, or climate); ii) specific infective agents (as worms, bacteria,or viruses); iii) inherent defects of the organism (as geneticanomalies); and/or iv) combinations of these factors

The terms “reduce,” “inhibit,” “diminish,” “suppress,” “decrease,”“prevent” and grammatical equivalents thereof (including, but notlimited to, “lower,” “smaller,” etc.) when used in reference to theexpression of any symptom in an untreated subject relative to a treatedsubject, mean that the quantity and/or magnitude of the symptoms in thetreated subject is lower than in the untreated subject by any amountthat is recognized as clinically relevant by any medically trainedpersonnel. In one embodiment, the quantity and/or magnitude of thesymptoms in the treated subject is at least 10% lower than, at least 25%lower than, at least 50% lower than, at least 75% lower than, and/or atleast 90% lower than the quantity and/or magnitude of the symptoms inthe untreated subject.

The term “migration” or “migrate” or “migrating” as used herein, refersto any movement of a cell (e.g., a T cell) in the direction of acompromised target tissue. Such migration may be accompanied by astimulation from a gradient of chemoattractant and/or chemotacticfactors (i.e., for example, lysophosphatidic acid) released by whiteblood cells.

The term “chemoattractant factor” as used herein, refers to any compoundand/or molecule that induces movement of chemotactic cells in thedirection of its highest concentration. For example, a chemoattractantfactor may include, but is not limited to, CCL22 and/or vasoactiveintestinal peptide (VIP).

The term “chemotactic cells” as used herein, refers to any biologicalcell exhibiting chemotaxis, wherein the chemotactic cells direct theirmovements according to certain chemicals in their environment.

The term “drug” or “compound” as used herein, refers to anypharmacologically active substance capable of being administered whichachieves a desired effect. Drugs or compounds can be synthetic ornaturally occurring, non-peptide, proteins or peptides, oligonucleotidesor nucleotides, polysaccharides or sugars.

The term “administered” or “administering” as used herein, refers to anymethod of providing a composition to a patient such that the compositionhas its intended effect on the patient. An exemplary method ofadministering is by a direct mechanism such as, local tissueadministration (i.e., for example, extravascular placement), oralingestion, transdermal patch, topical, inhalation, suppository etc.

The term “patient” as used herein, is a human or animal and need not behospitalized. For example, out-patients, persons in nursing homes are“patients.” A patient may comprise any age of a human or non-humananimal and therefore includes both adult and juveniles (i.e., children).It is not intended that the term “patient” connote a need for medicaltreatment, therefore, a patient may voluntarily or involuntarily be partof experimentation whether clinical or in support of basic sciencestudies.

The term “protein” as used herein, refers to any of numerous naturallyoccurring extremely complex substances (as an enzyme or antibody) thatcomprise a polymer of amino acid residues joined by peptide bonds,containing the elements carbon, hydrogen, nitrogen, oxygen, and usuallysulfur. In general, a protein comprises amino acids having an order ofmagnitude within the hundreds.

The term “peptide” as used herein, refers to any of various amides thatare derived from two or more amino acids by combination of the aminogroup of one acid with the carboxyl group of another and are usuallyobtained by partial hydrolysis of proteins. In general, a peptidecomprises amino acids having an order of magnitude with the tens.

The term “pharmaceutically” or “pharmacologically acceptable” as usedherein, refer to molecular entities and compositions that do not produceadverse, allergic, or other untoward reactions when administered to ananimal or a human.

The term, “pharmaceutically acceptable carrier”, as used herein,includes any and all solvents, or a dispersion medium including, but notlimited to, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils, coatings, isotonic and absorption delayingagents, liposome, commercially available cleansers, and the like.Supplementary bioactive ingredients also can be incorporated into suchcarriers.

The term, “purified” or “isolated” as used herein, may refer to apeptide composition that has been subjected to treatment (i.e., forexample, fractionation) to remove various other components, and whichcomposition substantially retains its expressed biological activity.Where the term “substantially purified” is used, this designation willrefer to a composition in which the protein or peptide forms the majorcomponent of the composition, such as constituting about 50%, about 60%,about 70%, about 80%, about 90%, about 95% or more of the composition(i.e., for example, weight/weight and/or weight/volume). The term“purified to homogeneity” is used to include compositions that have beenpurified to ‘apparent homogeneity” such that there is single proteinspecies (i.e., for example, based upon SDS-PAGE or HPLC analysis). Apurified composition is not intended to mean that some trace impuritiesmay remain.

The term “substantially purified” as used herein, refers to molecules,either nucleic or amino acid sequences, that are removed from theirnatural environment, isolated or separated, and are at least 60% free,preferably 75% free, and more preferably 90% free from other componentswith which they are naturally associated. An “isolated polynucleotide”is therefore a substantially purified polynucleotide.

The term “small organic molecule” as used herein, refers to any moleculeof a size comparable to those organic molecules generally used inpharmaceuticals. The term excludes biological macromolecules (e.g.,proteins, nucleic acids, etc.). Preferred small organic molecules rangein size from approximately 10 Da up to about 5000 Da, more preferably upto 2000 Da, and most preferably up to about 1000 Da.

The term “immunologically active” defines the capability of a natural,recombinant or synthetic peptide, or any oligopeptide thereof, to inducea specific immune response in appropriate animals or cells and/or tobind with specific antibodies.

The term “artificial particle” or “artificial construct” as used herein,refers to any fabricated degradable polymer construct that is capable ofemulsification to encapsulate soluble factors, wherein the polymer maybe functionalized to attached surface bound factors. The constructs maythen manipulate native regulatory cells to induce immunologicaltolerance.

The term “artificial presenting cell” as used herein, refers to anyartificial particle or artificial construct that further comprises animmunological synapse.

The term “immunological synapse’ as used herein, refers to any specific,non-random, arrangement of biological compounds (i.e., for example,antibodies or soluble proteins) on a biological antigen presenting cellthat is complementary to a contacting region on a target cell (i.e., forexample, a lymphocyte). For example, an antigen presenting cell maycomprise an immunological synapse that resembles a “bull's eye” and hasa complementary contact region on a regulatory T cell (T_(reg) cell).

The term “artificial dendritic cell” or “artificial APC” as used herein,refers to any fabricated degradable, artificial presenting cell usingmicroparticle-based controlled release technology capable of interactingwith a regulatory T cell. For example, cell-sized PLGA microparticlesmay be used to encapsulate soluble factors that are capable of inducingregulatory T cells to induce immunosuppression. For investigationalstudies, the soluble factors may be labeled with monoclonal antibodies(i.e., for example, fluorescent labels).

The term “artificial osteoblast cell” as used herein, refers to anyfabricated degradable microparticle having controlled release technologycapable of interacting with an osteoclast cell. For example, cell-sizedPLGA microparticles may be used to encapsulate soluble factors that arecapable of inducing osteoclasts to initiate bone healing. Forinvestigational studies, the soluble factors may be labeled withmonoclonal antibodies (i.e., for example, fluorescent labels).

The term “artificial osteoclast cell” as used herein, refers to anyfabricated degradable microparticle having controlled release technologycapable of interacting with an osteoblast cell. For example, cell-sizedPLGA microparticles may be used to encapsulate soluble factors that arecapable of inducing osteoblasts to modulate bone healing. Forinvestigational studies, the soluble factors may be labeled withmonoclonal antibodies (i.e., for example, fluorescent labels).

The term “soluble regulatory T cell stimulating factors” as used herein,refers to any biological agent (i.e., for example, a protein, hormone,drug etc.) capable of interacting with T_(reg) cells that may becomeencapsulated within a polymer-based microparticle (i.e., for example, anartificial antigen presenting cell) and undergo controlled releaseduring the degradation of the microparticle. Such soluble factors maybe, for example, CCL22, IL2, or TGFβ.

The term “soluble osteoclast signaling factor” as used herein, refers toany biological agent (i.e., for example, a protein, hormone, drug etc.)capable of interacting with osteoclast cells that may becomeencapsulated within a polymer-based microparticle (i.e., for example, anartificial osteoblast cell) an undergo controlled release during thedegradation of the microparticle. Such soluble factors may be forexample, MIM-1.

The term, “surface bound regulatory T cell stimulating factors” as usedherein, refers to any biological agent (i.e., for example, a protein,hormone, drug etc.) capable of interacting with T_(reg) cells, that areattached to the surface of a polymer-based microparticle (i.e., forexample, an artificial antigen presenting cell) and are not releasedduring the degradation of the microparticle. Such surface factors maybe, for example, CD3 antibody, CD28 antibody, or adhesion molecules.

The term, “surface bound osteoclast signaling factors” as used herein,refers to any biological agent (i.e., for example, a protein, hormone,drug etc.) capable of interacting with osteoclast cells that areattached to the surface of a polymer-based microparticle (i.e., forexample, an artificial antigen presenting cell) and are not releasedduring the degradation of the microparticle. Such surface factors maybe, for example, RANK-L.

The term “biomimetic” as used herein, refers to any artificial orsynthetic construct that has a similar biological effect as a nativebiological compound. Such similar effects may be due to similarities issize, shape, or specific (i.e., non-random) display of receptors,antibodies, or other active biological compounds. Such shapes include,but are not limited to, a circle, a square, a rectangle, a triangle, atrapezoid, a hexagon, an octagon, or a tetrahedron.

The term “biocompatible” as used herein, refers to any material does notelicit a substantial detrimental response in the host. There is alwaysconcern, when a foreign object is introduced into a living body, thatthe object will induce an immune reaction, such as an inflammatoryresponse that will have negative effects on the host. In the context ofthis invention, biocompatibility is evaluated according to theapplication for which it was designed: for example; a bandage isregarded a biocompatible with the skin, whereas an implanted medicaldevice is regarded as biocompatible with the internal tissues of thebody. Preferably, biocompatible materials include, but are not limitedto, biodegradable and biostable materials.

The term “biodegradable” as used herein, refers to any material that canbe acted upon biochemically by living cells or organisms, or processesthereof, including water, and broken down into lower molecular weightproducts such that the molecular structure has been altered.

The term “controlled release” as used herein, refers to the escape ofany attached or encapsulated factor at a predetermined rate. Forexample, a controlled release of a factor may occur resulting from thepredictable biodegradation of a polymer particle (i.e., for example, anartificial antigen presenting cell, a microparticle and/or amicrosphere). The rate of biodegradation may be predetermined byaltering the polymer composition and/or ratio's comprising the particle.Consequently, the controlled release may be short term or the controlledrelease may be long term. In one embodiment, the short term release isbetween 30 minutes-1 hour. In one embodiment, the short term release isbetween 1 hour-3 hours. In one embodiment, the short term release isbetween 3 hours-10 hours. In one embodiment, the short term release isbetween 10 hours-24 hours. In one embodiment, the long term release isbetween 24 hours-36 hours. In one embodiment, the long term release isbetween 3 days 7 days. In one embodiment, the long term release isbetween 7 days-1 month. In one embodiment, the long term release isbetween 1 month-6 months. In one embodiment, the long term release isbetween 6 months-1 year. In one embodiment, the long term release is atleast one year.

The term “emulsification” as used herein, refers to any process thatmixes together at least two compounds that are not naturally miscibletogether. For example, an emulsification process may utilize agents(i.e., emulsifiers) that facilitate the co-solubility of immisciblecompounds. For example, emulsification may include methods that usetechniques including, but not limited to, precipitation, emulsions, meltcasting, spray drying, crystallization, shearing, or milling.

The term “functionalized polymer” as used herein, refers to any polymerwhere the terminal moiety has been chemically altered to facilitate theattachment of other chemicals and or compounds (i.e., for example,proteins, antibodies, hormones, drugs, etc.). For example, the terminalmoiety may include, but is not limited to, a carboxylic acid, an amine,a sulfide, a hydroxyl etc.

The term “immobilization” as used herein, refers a state where abiological compound is fixed in position as a complex and is unable tomove without the movement of the complex.

The term “covalent bond” as used herein, refers to a close associationbetween at least two atoms, wherein the atoms share electrons inspecific atomic orbital paths.

The term “non-covalent bond” as used herein, refers to a closeassociation between at least two atoms, wherein the atoms do not shareelectrons.

The term “tissue transplant” as used herein, refers to any replacementof a tissue and/or organ within an individual with a similar tissueand/or organ from a different individual. In some cases, the individualsare from the same species. In other cases, the individuals are fromdifferent species.

The term “immunological tolerance” as used herein, refers to anymodification of the immune system wherein specific antibodies may not beproduced, but the immune system remains responsive to other antigens.For example, specific immune related cells including, but not limitedto, T_(reg) cells, osteoclasts, and/or osteoblasts may be stimulated toinduce immunosuppression. Such “immunological tolerance” may also becapable of controlling autoimmune diseases including, but not limitedto, arthritis, Type I diabetes. Such “immunological tolerance” may alsobe capable of controlling inflammatory diseases including, but notlimited to, periodontal disease such as periodontitis.

The term, “supramolecular activation clusters (SMACs)” as used herein,refer to a topologically complex, and discretely organized, arrangementof antigen presenting cell surface proteins and/or soluble proteins.SMACs may be comprised of a plurality of regions having localizeddensity of specific surface proteins and/or soluble proteins (i.e., forexample, central SMAC (cSMAC), peripheral SMAC (pSMAC), or distal SMAC(dSMAC). In general, the surface proteins comprise cell surfaceantibodies and/or receptors, while the soluble proteins comprise ligandsdirected to activating certain lymphocytes (i.e., for example, aregulatory T cell).

The term “apical region” as used herein, refers to the top of a sphereand spreading circumferentially from between approximately 1 degree to30 degrees from a polar axis. In one embodiment, the apical region mayspread circumferentially from between approximately 5 degrees to 20degrees from a polar axis. In one embodiment, the apical region mayspread circumferentially from between approximately 10 degrees to 15degrees from a polar axis.

The term “tropical region” as used herein, refers to a circumferentialregion of a sphere located between approximately 30-70 degrees from apolar axis. In one embodiment, the tropical region may spreadcircumferentially from between approximately 35 degrees to 60 degreesfrom a polar axis. In one embodiment, the tropical region may spreadcircumferentially from between approximately 40 degrees to 50 degreesfrom a polar axis.

The term “equatorial region” as used herein, refers to a circumferentialregion of a sphere located between approximately 70-90 degrees from apolar axis. In one embodiment, the equatorial region may spreadcircumferentially from between approximately 75 degrees to 85 degreesfrom a polar axis. In one embodiment, the equatorial region may spreadcircumferentially from between approximately 87 degrees to 93 degreesfrom a polar axis.

The term “random” as used herein, refers to a stochastic arrangement ofobjects formed without definite aim, purpose, method, or adherence to aprior arrangement; or in a haphazard way.

The term “non-random” as used herein, refers to a non-stochasticarrangement of objects with a definite aim, purpose, method and adheresto a prior conceived pattern.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides exemplary data showing a T_(reg) proliferative doseresponse induced by degradable particles with randomly attached CD3antibodies and CD28 antibodies (ratio=1:1 or 1:4 cells/particle).Proliferation was measured by tritiated thymidine incorporation.Controls are equivalent amounts of soluble antibody (control). Inset:FITC labeled CD3 and CD28 antibodies for visualization),

FIG. 2 presents an illustrative embodiment showing how regulatory Tcells (T_(egs)) are attracted to: (A) a tumor; or (B) an artificialpresenting cell (i.e., for example, a synthetic dendritic cell) near thesurface of an allograft.

FIG. 3 shows a fluorescent image of a representative “bull's eye”immunological synapse orientation from a biological dendritic cell.Grakoui et al., Science 285:221 (1999).

FIG. 4A illustrates one embodiment of a method for fabricatingartificial presenting cell construct patterns that mimic a cellularimmunological synapse (IS).

FIG. 4B presents an exemplary fluorescent photomicrograph image of asynthetic dendritic cell comprising a central supramolecular activationcomplex (cSMAC) region (green/yellow) and a peripheral supramolecularactivation complex (pSMAC) region. See, Top Image arrows, respectively.Scanning Electron Microscopic backscatter imaging shows a bright, andintentionally over-labeled, cSMAC region (See, Bottom Image, whitearrow=gold-bead labeled protein).

FIG. 5A presents an illustration of one embodiment for the fabricationof polymer microparticles via a double-emulsion/solvent evaporationtechnique.

FIG. 5B presents an exemplary freeze fracture scanning electronmicrograph of a polymer microparticle created by the technique shown inFIG. 5A demonstrating an inner aqueous phase.

FIG. 6 illustrates standard carbodiimide chemistry to create an aminereactive site on the surface of PLGA microparticles starting with acarboxylic acid generated from the degradation of ester bonds.

FIG. 7 presents one embodiment to label functionalized PLGA polymersusing a novel compound (i.e., for example, 2-pyridyldithioethylamine) toconvert a carboxylic acid group into a thiol reactive group (See, middlepanel). A subsequent cleavage reaction liberates a detectable productupon conjugation which can be used to determine the extent of surfacelabeling (see, right panel).

FIG. 8 illustrates one embodiment of an in vitro chemotaxis assay.Microparticles containing chemokines are placed in the bottom of atranswell filter plate with T_(reg) cells isolated from a transgenicB6/GFP+mouse on top of the opaque filter (left). After a time, chemokineis released and T_(reg) cells migrate through the filter and up theconcentration gradient of the chemokine (right). Fluorescence readingsare taken with a top and bottom detector plate reader to determine theextent of Treg cell chemotaxis.

FIG. 9A presents exemplary data showing a release profile of CCL22 fromporous microparticles showing an approximate linear release for overtwenty (20) days.

FIG. 9B presents exemplary data showing the successful creation ofmicroparticles encapsulating CCL22 as used in FIG. 9A. The top portionshows a representative image of an intact microparticle, indicating theparticle's structural integrity and confirming the size. The bottomportion shows a representative image of a fractured particle confirmingthe porous nature of the particle created by the double emulsionencapsulation process.

FIG. 10 presents exemplary data showing CCL22 labeling (red) using themicroparticles in accordance with FIG. 9.

FIG. 11 presents an illustration of one embodiment comparing healthy gumtissue (left side) with gum tissue affected by periodontal disease(right side).

FIGS. 12A and 12B present exemplary data showing the effect ofartificial antigen presenting cells (e.g., a polymer microparticle)encapsulating CCL-22 on periodontal disease in a mouse model. AA:infected, untreated mice. LP: infected, aAPC-CCL22 treated mice; CP:infected, aAPC-only mice.

FIG. 13A presents an illustration representing a hypothesized couplinginteraction for osteoclast stimulation. For example, osteoclasts may berecruited by MCSF and activated by surface-bound RANK-L and/or OSCAR-L.

FIG. 13B presents an illustration of an artificial osteoblast cell thatmimics the coupling interactions shown in FIG. 13A.

FIG. 14A presents an illustration representing a hypothesized couplinginteraction for osteoblast stimulation. For example, osteoblasts arerecruited by MIM1 and activated by surface-bound TGF-beta and EphrinB2(BMP2).

FIG. 14B presents an illustration of an artificial osteoclast cell thatmimics the coupling interactions shown in FIG. 14A.

FIG. 15 presents representative scanning micrographs of CCL22-containingpolymer microparticles.

FIG. 15A shows the external porous structure of a polymer microparticle.

FIG. 15B shows the internal structure of a polymer microparticledisplaying an inner aqueous phase created by a water-in-oil emulsion.

FIG. 16 presents exemplary data showing release kinetics of CCL22 frompolymer microparticles as measured in physiological buffered saline(PBS). Error Bars=Standard Deviations; n=3.

FIG. 17 presents exemplary data showing a representative volume of anaveraged sized distribution of polymer microparticles comprising CCL22determined by average volume diameter measurements. Error Bars ═StandardError of the Mean; n=6.

FIG. 18 presents exemplary data showing CCR4 expression and in vitrotranswell migration assay.

FIG. 18A: Histograms depicting the absence of CCR4 expression on naïveCD4+ T cells but its presence on activated CD4+ T cells, as determinedby flow cytometry.

FIG. 18B: Transwell migration assays show significantly greatermigration of activated regulatory and effector CD4+ T cells whencompared to respective populations of naïve cells at differentconcentrations of CCL22. (* p<0.05, one-tailed Students T test)

FIG. 18C: Expression of CCL22 is enhanced in CD4+ cells in the presenceFoxP3 in comparison to the absence of FoxP3.

FIG. 19 presents exemplary data showing an in vivo migration of Tregstowards polymer microparticles comprising CCL22.

FIG. 19A shows a representative schematic describing one experimentaldesign for live animal imaging Luc+ Treg indicates a Treg populationisolated from transgenic mice constitutively expressing the luciferasegene.

FIG. 19B presents exemplary data showing representative fluorescence andluminescence images obtained at day 7 post injection, fluorescenceimages were used to outline areas of particle localization (i.e., forexample, with Pro Living Image® 2.60.1) and these outlines weresuper-imposed onto luminescence images taken with the mouse in the exactsame position to demonstrate co-localization of Tregs and CCL22microparticles.

FIG. 19C presents exemplary data showing kinetics of Treg migrationtowards the injection sites; average radiance measurements obtained fromthe luminescence images are displayed as a ratio of CCL22 microparticles(CCL22MP) to Blank microparticles (BlankMP). Error Bars=StandardDeviation; n=3.

FIG. 20 presents exemplary data of non-invasive live animal imaging. Theleft limb was injected with BlankMP-680 and the right limb was injectedwith CCL22MP-680. Representative fluorescence (left) and luminescence(right) images taken from a single mouse at different time pointsfollowing injection. Days 4 & 7: Significant co-localization of Tregwith CCL22 microparticles. Days 2 and 9: Very little or noco-localization of Treg with CCL22 microparticles. Values represent thetotal flux (photons/sec) of fluorescence or luminescence intensities.ROI (regions of interest) were drawn automatically using Igor Pro LivingImage® 2.60.1. Influx of Treg only towards CCL22MP was observed overmultiple experimental cycles (n=6). Over time it was observed that Treglocalized in mucosal tissues such as the gut and the lung, which secreteCCL22 endogenously. Endogenous Treg migration to the hind limbs wasnegligible and the cells that migrated to these sites were specificallytowards CCL22MP.

FIG. 21 presents one method of preparing polymer microparticlescomprising a double emulsion and evaporation.

FIG. 22 presents exemplary data showing mRNA expression of markers inperiodontal tissues resected from diseased mice:

FIG. 22A: FoxP3: a marker for Tregs.

FIG. 22B: Anti-inflammatory, pro-regenerative cytokine IL-10.

FIG. 22C: Bone resorbing cell (osteoclast) activation factor RANKL.

FIG. 22D: Endogenous CCL-22 (Treg recruiting) chemokine.

FIG. 23 presents exemplary data showing abrogation of alveolar boneresorption in a mouse model of periodontitis. Dissecting microscopeimages of resected maxilla were mechanically de-fleshed and soaked indispase overnight. Dotted-red boxes outline large differences inalveolar bone levels between the internal control and CCL-22 MP treated.

FIG. 23A: Representative image from mice that received BlankMPs A RIGHTin the right maxilla and no treatment (internal control) A LEFT in theleft maxilla.

FIG. 23B: Representative image from a mouse that received CCL-22 MPs BRIGHT in the right maxilla and no treatment (internal control) B LEFT inthe left maxilla.

FIG. 23C: Area measurements (CEJ-ABC) for the right maxilla of mice fromeach treatment and control group. *p-value<0.05

FIG. 24 presents exemplary data showing tumor regression after inductionof Treg cells. Transgenic luciferase expressing lewis lung carcinomacells (derived from C57B1/6 mice) were injected into FVB mice 4 dayspost particle injections. Quantitative live animal imaging was performedsubsequently at regular time intervals showing qualitative data obtainedat 2 different time points, suggesting that the CCL22 microparticles areable to slow down rejection of cellular allo-transplants.

FIG. 25 presents tumor regression kinetic data from the experimentdemonstrated in FIG. 24. * Indicates p<0.05; Wilcoxon rank-sum test fornull hypothesis that normalized luminescence for CCL22MP is same as thatof BlankMP or bolus CCL22. # p≦0.05 (comparison between CCL22MP andBlankMP only). n=11 BlankMP and CCL22MP (n=5 for bolus CCL22).

FIG. 26 presents a representative schematic of a proposed mechanism forvasoactive intestinal peptide (VIP) induction of regulatory T cells. Forexample, by a direct activation of CD4+CD25− naïve T cells to inducibleTregs. Alternatively by an activation of tolerogenic dendritic cellswhich then promote inducible Treg generation through activation ofCD8+CD25− naïve T cells.

FIG. 27 presents exemplary data showing VIP release from VIPMPs havingrelease profiles predicted by a computer algorithm presented herein.

FIG. 27A: VIP release profiles from predicted linear release VIPMPs(diamonds) and predicted multi-bolus release VIPMPs (squares). Standarddeviations are represented by bars at each data point.

FIG. 27B. Comparison of the algorithmic model release prediction (redline), observed VIP release (blue diamonds) from the predicted linearrelease VIPMPs to the desired profile model input (dashed line). Theanomalous initial release burst was ignored for this comparison.

FIG. 28 presents exemplary data showing the percent of FoxP3+ CD4+T-cells which were recruited through the transwells by DCs, followingtreatment with LPS and addition of soluble VIP (2.5×10⁻⁸M), blankmicroparticles releasates, or VIP microparticle releasates (estimated tobe at 2.5×10⁻⁸ M from previous release profiles). Percents werenormalized by the group which did not receive LPS. Upper Graph: DCprecursors treated with IL-4 and GM-CSF. Lower Graph: DC precursorscultured with 5×GM-CSF.

FIG. 29 presents exemplary data showing linear and volumetricmeasurements of alveolar bone loss using micro-CT imaging as shown inPark et al., J Periodontol. 78(2):273-281 (2007).

FIG. 30 presents an exemplary micro-CT analysis performed on mice havingperiodontitis after receiving VIPMP treatment.

FIG. 31 presents exemplary scanning electron microscope images of VIPmicroparticles comprising a 12.6 kDa PLGA polymer. Nonporous (A) andporous (B) VIP microparticles had average diameters of 16.7 μm and 17.714.3 μm, respectively.

FIG. 32 presents exemplary VIP release profiles from PLGA VIPmicroparticles in PBS consisting of either: a 4.2 kDa polymer (bluediamond); a 12.6 kDa polymer (red square); or a 100 kDa polymer (greentriangle). Standard deviations are represented by bars at each datapoint.

FIG. 33 presents an illustrative comparison of VIP release between acomputer modeling prediction (red line) with actual observed VIP releasedata (blue diamonds) for individual PLGA microparticles consisting of4.2 kDa polymers (upper panel), 12.6 kDa polymers (middle panel), or 100kDa polymers (lower panel).

FIG. 34 presents exemplary data of a VIP release profile frommicroparticles consisting of 12.6 kDa PLGA polymers showing a smallermagnitude of initial burst than the data shown in FIG. 33.

FIG. 35 presents exemplary data comparing actual VIP release profiles inPBS from predicted linear and/or multi-bolus VIPMPs.

FIG. 35A: VIP release profiles from either predicted extended linearVIPMPs (blue diamonds) or predicted multi-bolus VIPMPs (red squares).Standard deviations are represented by bars at each data point.

FIG. 35B: Comparison of computer algorithm model prediction (red line),observed VIP release from predicted linear VIPMPs (blue diamonds) andthe desired profile computer model input (dashed line). The anomalousinitial burst release was ignored for this comparison.

FIG. 36 presents exemplary data showing VIP release profiles fromnonporous VIP 12.6 kDa polymer MPs (blue diamonds) and porous VIP 12.6kDa polymer MPs (red squares) in PBS. Standard deviations arerepresented by bars at each data point.

FIG. 37 presents exemplary data showing cumulative VIP release profilesfrom 12.6 kDa polymer VIPMPs either with PEG (red squares) or withoutPEG (blue diamonds) in PBS. Standard deviations are represented by barsat each data point.

FIG. 38 presents an illustrative comparison of VIP release profiles fromVIP 100 kDa PLGA polymer MPs (blue diamonds) and VIP 12.6 kDa PLGApolymer MPs with PEG (red squares) in the inner aqueous phase. PEGappears to minimize the VIP 12.6 kDa polymer MP initial burst as seenabove, and provide a near-linear release of VIP similar to that of VIP100 kDa polymer MPs.

FIG. 39 presents exemplary data of CCL-22 production by DCs measured at7 hours (upper panel), 24 hours (middle panel), and 48 hours (lowerpanel) following treatment. mDC Control group: no LPS or drug. SolubleVIP group: 10⁻⁶ M soluble VIP. Blank MPs: 0.0556 mg/mL of nonporousempty MPs; and VIP MPs 0.0556 mg/mL of nonporous VIPMPs.

FIG. 40 presents exemplary data showing a reduction of alveolar boneloss in mice infected with periodontal disease that were treated withVIPMPs.

FIG. 40A: Dissecting microscope images of bone loss between the alveolarbone crest and cementoenamel junction for mice treated with BlankMPs,CCL22MPs, or VIPMPs.

FIG. 40B: Quantitative bone loss area for sham, BlankMPs, VIPMPs, andCCL22MPs. Mice treated with VIPMPs or CCL22MPs showed significantly lessbone loss than the BlankMPs. p<0.021 and p<0.002, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to the treatment of immunologicaldisorders, immunological diseases and/or transplantation rejectionreactions. For example, certain embodiments of the invention result inthe regulation of blood cells including, but not limited to, naïve bloodcells and/or white blood cells (e.g., T cells and/or B cells) controlledby vasoactive intestinal peptide (VIP). In particular, the inventioncontemplates methods of creating VIP-induced regulatory T cells bycontrolled VIP release from custom designed microparticles.

The absence of regulatory T cells (Treg) may underlie immunologicaldisorders including, but not limited to, autoimmunity, dermatitis,periodontitis and/or transplant rejection Enhancing local numbers ofTreg through in situ Treg expansion or induction is contemplated hereinas a treatment option for these disorders. Current methods for in vivoTreg expansion are not Treg-cell specific and are associated with manyadverse side-effects. The data presented herein provides in vitrotesting of a Treg-inducing microparticle populations providing apredictable controlled release of vasoactive intestinal peptide (VIP).These controlled release microparticles are also capable of inducingFoxP3+ Treg in human cells in vitro suggesting that these microparticlesmay be clinically useful for in vivo Treg induction and expansiontherapy.

Current techniques for regulatory T cell induction can only be used invitro as they rely on the use of soluble mediators. In some embodiments,the present invention contemplates compositions and methods to deliver Tcell induction soluble factors in vivo in a sustained, predictable andcontrolled manner.

In one embodiment, the present invention contemplates methods forevaluating the therapeutic benefits of vasoactive intestinal peptide(VIP) in treating periodontitis. Although it is not necessary tounderstand the mechanism of an invention, custom microparticlecompositions having specific VIP release profiles were predicted bycomputer modeling and validated by formulating microparticles havingspecific ratios of polymers having different molecular weights. Forexample, in one embodiment, custom microparticles were formulated using4.2 kDa PLGA, 12.6 kDa PLGA, and 100 kDa PLGA. In other embodiments, thecustom microparticle further comprises an inner aqueous phase comprisingsodium chloride (NaCl) and/or polyethylene glycol (PEG). The datapresented herein demonstrates that the released VIP from custommicroparticles was bioactive and caused dendritic cells (DCs) to producemore CCL22 than DCs treated with blank particles after 7 and 24 hours ofstimulation (p<0.05). The data further shows that DCs treated with VIPmicroparticle releasates recruited higher percentages of FoxP3+ T-cellsin in vitro chemotaxis studies. In vivo mouse model testing indicatedthat VIP microparticles have significant therapeutic potential to treatperiodontal disease by reducing the bone loss in infected mice relativeto Blank microparticles given to the control group (p<0.02).

The present invention is also related to the field of inducingimmunological tolerance by specifically manipulating immune-relatedcells. Such immunological tolerance may be induced by providingbiomimetic artificial microparticles that bypass native immunologicalcells. For example, biomimetic artificial particle compositions arecontemplated which comprise soluble factors that activate specificimmune-related blood cells, including, but not limited to, macrophagesand/or monocytes. Such factors may comprise chemoattractant factors.These biomimetic artificial microparticles may further present aspecific biomimetic surface pattern that results in a targeted cellresponse such that the particles represent artificial presenting cells.

The present invention contemplates artificial microparticles such that asynthetic construct may be capable of presenting the samereleased/secreted and/or chemically-bound/plasma membrane-boundcompounds (i.e., for example, soluble proteins, antibodies, receptors,hormones etc.) as actual living cells, and is not limited to modulatingthe immune system (i.e., for example, modulation of bone healing, tissueinflammation and/or tumor regression). The present inventioncontemplates various embodiments wherein these artificial microparticlesreplace biological cell-cell interactions within the processes of manybiological functions including, but not limited to, wound healing,restoration from a diseased state, or enhancement in normal function ofcellular processes.

In one embodiment, the present invention contemplates an investigationalplatform capable of testing the in vivo roles and efficiencies of bothsoluble secreted and surface bound T_(reg) cell factors. Such Treg cellfactors may include, but are not limited to, stimulatory factors,inducing factors and/or chemoattractant factors. In one embodiment, theinvestigational platform comprises an in vivo testing platform (i.e.,for example, an in vivo mouse model). In one embodiment, theinvestigational platform comprises an in vitro testing platform (i.e.,for example, a primary cell culture). These investigational platformsare capable of evaluating various combinations of membrane surfacefactors and/or soluble releasable factors that are attached to and/orencapsulated within, a biodegradable microparticle population thatstimulates T_(res) cells and induces allograft tolerance.

Although it is not necessary to understand the mechanism of aninvention, it is believed that these biodegradable microparticlepopulations may act as “artificial antigen presenting cells” thatcomprise a biomimetic immunological synapse that encapsulate andcontrollably release soluble factors for T_(reg) cell stimulation,T_(reg) cell proliferation, and T_(reg) cell chemotaxis. Hoffmann etal., Blood 104, 895-903 (2004); Green et al., Proc Natl Acad Sci USA100: 10878-10883 (2003); Curiel et al., Nat Med 10:942-949 (2004); andBelghith et al., Nat Med 9:1202-1208 (2003). Further, thesemicroparticle populations are capable of presenting surface-boundmonoclonal antibodies (mAb) specific for T_(reg) cell surface activationfactors.

I. Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) is a neuropeptide of twenty-eight(28) amino acids in length that was originally isolated from theintestine that has numerous biological and regulatory functions. Forexample, VIP down-regulates a variety of pro-inflammatory responses andup-regulates anti-inflammatory responses. Reports have suggested thatVIP may be useful in the treatment of experimental collagen-inducedarthritis, sepsis, Crohn's disease, and experimental autoimmuneencephalomyelitis (EAE) in mice. Delgado et al., (2001) “Vasoactiveintestinal peptide prevents experimental arthritis by downregulatingboth autoimmune and inflammatory components of the disease” Nat. Med.7:563-568; Fernandez-Martin et al., (2006) “Vasoactive intestinalpeptide induces regulatory T cells during experimental autoimmuneencephalomyelitis” Eur. J. Immunol. 36:318-326. Additionally, VIP mayshift the Th1-Th2 balance in the favor of Th2 by promoting Th2-typecytokine production and, at the same time, inhibit Th1 development andresponses. Gonzalez-Rey et al., (2007) “Vasoactive intestinal peptideand regulatory T-cell induction: a new mechanism and therapeuticpotential for immune homeostasis” Trends in Molecular Medicine13:241-251.

More recently, new mechanisms have been proposed that identify apotential role for VIP in inducing and recruiting regulatory T (Treg)cells for maintaining immune homeostasis. FIG. 26. Both natural andinducible regulatory T cells may be involved in immune tolerance andregulation. Natural Treg cells can develop in the thymus asCD4+CD25+FoxP3+ Tregs, and expand into the periphery. So-calledinducible Treg cells can be generated from CD4+CD25− and CD8+CD25− naïveT cells under certain stimulation patterns. Treg induction by VIP isbelieved to occur in two primary pathways. First, VIP may directlyactivate naïve CD4+CD25− T cells into a regulatory phenotype. Secondly,VIP may steer immature dendritic cells (DCs) into a tolerogenicphenotype. Such tolerogenic DCs can then induce CD4+ and CD8+ naïve Tcells toward a regulatory phenotype (inducible Tregs). Finally, VIP caninduce tolerogenic dendritic cells to produce CCL22. CCL22 is achemokine reported to be involved in the recruitment of regulatory Tcells. Pozo et al., (2007) “Tuning immune tolerance with vasoactiveintestinal peptide: A new therapeutic approach for immune disorders”Peptides 28:1833-1846; and Delgado et al., (2004) “VIP/PACAPpreferentially attract Th2 effectors through differential regulation ofchemokine production by dendritic cells” FASEB J 18:1453-1455.

One difficulty in VIP-based therapies is the short half-life of VIP inthe body. One approach to address this challenge is to develop VIPanalogs which are bioactive but have improved metabolic stability.Misaka et al., “Inhalable powder formulation of a stabilized vasoactiveintestinal peptide (VIP) derivative: Anti-inflammatory effect inexperimental asthmatic rats” Peptides (2010) 31:72-78. Another approachis to protect the VIP by encapsulation into sterically stabledliposomes. However, the reports show limitations to these approaches.For example, VIP analogues only extend the half-life to 4 hours and theliposomes offer only short-term release, releasing 77-87% ofencapsulated VIP within the first two hours in vitro. Wernig et al.,(2008) “Depot formulation of vasoactive intestinal peptide byprotamine-based biodegradable nanoparticles” J of Controlled Release130:192-198; and Misaka et al., (2010) “Inhalable powder formulation ofa stabilized vasoactive intestinal peptide (VIP) derivative:Anti-inflammatory effect in experimental asthmatic rats” Peptides31:72-78. As contemplated herein, microparticles which deliver anextended release profile solves the above problems of conventionalVIP-based therapy platforms.

The data presented herein describes an alternative method of quantifyingVIP release using microparticles composed of 12.6 kDa PLGA. The releasedVIP was detected using a commercially available VIP Enzyme Immunoassaykit (Phoenix Pharmaceuticals). Dendritic cell (DC) cultures were testedto determine the bioactivity of VIP released from these microparticles.The results suggest that VIP microparticles have a significanttherapeutic potential as a treatment for periodontitis.

In one embodiment, the present invention contemplates a compositioncomprising a VIPMP formulation having a polymer composition predicted bymathematical analysis to provide a pre-determined release profile.

II. Regulatory T Cells

A. Immunological Role And Function

Over the past two decades, regulatory T cells (Treg) have beenidentified as one component of the mammalian immune system. Sakaguchi etal., “Immunologic tolerance maintained by CD25+ CD4+ regulatory T cells:their common role in controlling autoimmunity, tumor immunity, andtransplantation tolerance” Immunol Rev. (2001) 182:18-32; Sakaguchi etal., “Regulatory T cells and immune tolerance” Cell (2008) 133:775-787;Campbell et al., “Phenotypical and functional specialization of FOXP3+regulatory T cells” Nat Rev Immunol (2011) 11:119-130; and Bour-Jordanet al., “Regulating the regulators: costimulatory signals control thehomeostasis and function of regulatory T cells” (2009) 229:41-66. Onecommonly described, widely studied, and abundant regulatory T cells inthe body are those that express CD4, CD25, and/or FoxP3. These CD4+CD25+ FoxP3+ cells (Treg) are believed to play a role in suppressing theactivity of self-reactive immune cells and in re-establishinghomeostasis following infection. Sakaguchi et al., “Immunologicself-tolerance maintained by activated T cells expressing IL-2 receptoralpha-chains (CD25). Breakdown of a single mechanism of self-tolerancecauses various autoimmune diseases” J Immunol. (1995) 155:1151-1164;Hori et al., “Control of regulatory T cell development by thetranscription factor Foxp3” Science (2003) 299:1057-1061; Fontenot etal., “Foxp3 programs the development and function of CD4+ CD25+regulatory T cells” Nat. Immunol. (2003) 4:330-336; and Khattri et al.,“An essential role for Scurfin in CD4+CD25+ T regulatory cells” Nat.Immunol. (2003) 4:337-342.

B. Clinical Applications

Treg proliferation has been reported to suppress diverse inflammatorydiseases such as: i) autoimmunity (de Kleer et al., “CD4+CD25 brightregulatory T cells actively regulate inflammation in the joints ofpatients with the remitting form of juvenile idiopathic arthritis” JImmunol. (2004) 172:6435-6443: ii) transplant rejection (Raimondi etal., “Mammalian target of rapamycin inhibition and alloantigen-specificregulatory T cells synergize to promote long-term graft survival inimmunocompetent recipients” J Immunol (2010) 184:624-636; and Lee etal., “Recruitment of Foxp3+ T regulatory cells mediating allografttolerance depends on the CCR4 chemokine receptor” J Exp Med (2005)201:1037-1044; iii) dermatitis (Robinson et al., “Tregs and allergicdisease” J Clin Invest. (2004) 114:1389-1397; iv) psoriasis (Wang etal., “TGF-beta-dependent suppressive function of Tregs requireswild-type levels of CD18 in a mouse model of psoriasis” J Clin Invest.(2008) 118:2629-2639; Bovenschen et al., “Foxp3+ Regulatory T Cells ofPsoriasis Patients Easily Differentiate into IL-17A-Producing Cells andAre Found in Lesional Skin” J Invest Dermatol. (2011) 131:1853-1860; andv) periodontitis Garlet et al., “Actinobacillusactinomycetemcomitans-induced periodontal disease in mice: patterns ofcytokine, chemokine, and chemokine receptor expression and leukocytemigration” Microbes Infect. (2005) 7:738-747; Garlet et al., “RegulatoryT cells attenuate experimental periodontitis progression in mice” J ClinPeriodontol. (2010) 37:591-600.

Proliferation of Treg cell populations at local tissue sites has beenreported by various methods such as: i) ex vivo expansion of Treg cellsfollowed by a local administration or systemic re-infusion; and ii) invivo manipulation of immune cells that increases the Treg/Teff ratio.Riley et al., “Human T regulatory cell therapy: take a billion or so andcall me in the morning” Immunity (2009) 30:656-665; Safinia et al.,“Adoptive regulatory T cell therapy: challenges in clinicaltransplantation” Curr Opin Organ Transplant (2010) 15:427-434; andWieckiewicz et al., “T regulatory cells and the control of alloimmunity:from characterisation to clinical application” Curr Opin Immunol. (2010)22:662-668. Selective enhancement of Treg cell populations in vivo hasbeen reported using biologic therapies. For example, i) anti-IL-2monoclonal antibody (Webster et al., “In vivo expansion of Treg cellswith IL-2-mAb complexes: induction of resistance to EAE and long-termacceptance of islet allografts without immunosuppression” J Exp Med.(2009) 206:751-760; ii) superagonistic anti-CD28 monoclonal antibody(Beyersdorf et al., “Selective targeting of regulatory T cells with CD28superagonists allows effective therapy of experimental autoimmuneencephalomyelitis” J Exp Med. (2005) 202:445-455; and iii) agonisticanti-CD4 monoclonal antibody (Anonymous, “Deal watch: Boosting Tregs totarget autoimmune disease” Nat. Rev. Drug Discovery (2011) 10:566. Theseapproaches have specific disadvantages including, but not limited to, alimited understanding of the underlying mechanism of action and humansafety for clinical administration. In fact, phase I clinical trials ofthe superagonistic anti-CD28 monoclonal antibody (TGN1412) resulted insevere negative reactions (cytokine ‘storm’) in all 6 human subjects whoreceived the monoclonal antibody. Suntharalingam et al., “Cytokine stormin a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412” N EnglJ Med. (2006) 355:1018-1028. Establishment of a local immunosuppressiveenvironment that selectively favors Treg expansion has also been shownto increase Treg cell population numbers. An environment rich in IL-2,transforming growth factor-β1 (TGF-β) and rapamycin (an inhibitor of theserine-threonine kinase mammalian target of rapamycin; mTOR) has beenshown to favor Treg development, even under inflammatory conditions.Haxhinasto et al., “The AKT-mTOR axis regulates de novo differentiationof CD4+Foxp3+ cells” J Exp Med. (2008) 205:565-574; Kopf et al.,“Rapamycin inhibits differentiation of Th17 cells and promotesgeneration of FoxP3+ T regulatory cells” Int Immunopharmacol. (2007)7:1819-1824; and Cobbold et al., “Infectious tolerance via theconsumption of essential amino acids and mTOR signaling” Proc Natl AcadSci USA (2009) 106:12055-12060.

However, formulations providing a predictable continuous release ofthese factors in vivo, has proven difficult. This problem is solved asdescribed herein by microparticle compositions and methods showingcontrolled release profiles of Treg cell inducing compounds, such asvasoactive intestinal peptide can be mathematically predicted byadjustment of polymeric ratios comprising the microparticles. Thesepredicted microparticle compositions are expected to have a bettertherapeutic efficacy and safety than current antibody Treg inductionmodels, thereby having a superior clinical application to treat medicaldisorders and disease.

Therapies that enhance Treg numbers and function may have the potentialto suppress transplant rejection and autoimmunity. Clinical trials arecurrently underway to test cellular therapies involving Treg cells aspotential therapeutics for treating graft versus host disease. Hippen etal., “Generation and large-scale expansion of human inducible regulatoryT cells that suppress graft-versus-host disease” Am J. Transplant.(2011) 11:1148-1157; and Bronstein et al., “Infusion of ex vivo expandedT regulatory cells in adults transplanted with umbilical cord blood:safety profile and detection kinetics” Blood (2011) 117:1061-1070.

However, Treg-based cellular therapies face many challenges, whichinclude, but are not limited to: i) difficulties in isolating pure andhomogenous populations and large quantities of Treg from the blood; ii)inconsistent maintenance of the Treg phenotype and suppressive functionpost-proliferation; and iii) the need for GMP facilities. Riley et al.,“Human T regulatory cell therapy: take a billion or so and call me inthe morning” Immunity (2009) 30:656-665; Safinia et al., “Adoptiveregulatory T cell therapy: challenges in clinical transplantation” CurrOpin Organ Transplant. (2010) 15:427-434; and Wieckiewicz et al., “Tregulatory cells and the control of alloimmunity: from characterisationto clinical application” Curr Opin Immunol. (2010) 22:662-668. Hence,acellular therapies that can increase numbers and/or the suppressivepotency of Treg without the need for ex vivo culture represent an answerto a long unsolved problem in the art.

III. Induction Of Regulatory T Cell Phenotypes

Treg cells can be induced under a variety of in vitro and in vivoconditions. Treg cells may be induced under different conditions to havedistinct characteristics that distinguish them from each other and fromnaturally-occurring Treg cells. Identification of these distinctivecharacteristics provides insight into their potential use in treatinginflammatory disorders (such as autoimmunity and allergy) and transplantrejection, or in preventing tumor growth and metastasis. For example, ithas been demonstrated that RA can help to convert naïve T cells toinduced Treg cells (iTreg). These RA-iTreg are stable under inflammatoryconditions and have the potential to prevent inflammation in the gut dueprimarily to their ability to specifically migrate to the gut. However,such tissue-specific migration could potentially be a hindrance to usingthese types of iTreg cells to treat autoimmunity or transplant rejectionat other peripheral sites.

Regulatory T cells (Treg) may be involved in maintaining immunehomeostasis. Consequently, it is believed that Treg cell therapy mightbe useful to treat medical conditions including, but not limited to, avariety of immune mediated disorders. Current Treg cell-based clinicaltherapies have disadvantages including, but not limited to, obtaininginsufficient numbers of cells from peripheral blood for expansion andre-infusion. One alternative method to induce the formation of Tregcells from non-Treg cells has been reported that contacts non-Treg cellswith a soluble cytokine (e.g., TGF-β). However, this approach hasdisadvantages including, but not limited to, the fact that these methodsdo not induce stable Treg cells in sufficient number to be useful.Although it is not necessary to understand the mechanism of aninvention, it is believed that all-trans retinoic acid (RA) and/orrapamycin (RAPA) may aid in achieving a stable induced Treg (iTreg)phenotype. Even so, such iTreg phenotypes have not been characterized asto phenotype, function and/or migratory characteristics

In one embodiment, the present invention contemplates a method to inducea specific and stable phenotype and/or function of iTreg cells. In oneembodiment, the iTreg cells may be produced by contacting non-Treg cellswith a compound such as VIP.

Regulatory T (Treg) cell-based therapies are widely regarded aspromising treatment options for immunological diseases including, butnot limited to, autoimmunity and/or transplant rejection. Currently,several therapies involving the use of ex vivo expanded Treg cells arebeing tested in clinical trials. However, there are significant barriersto ex vivo Treg cell-based therapies, such as difficulty in isolatingpure populations of these rare cells and expanding them to sufficientlylarge numbers while maintaining their phenotype and function.Wieckiewicz et al., (2010) “T regulatory cells and the control ofalloimmunity: From characterisation to clinical application” Curr OpinImmunol 22: 662-668; Riley et al., (2009) “Human T Regulatory CellTherapy: Take a Billion or So and Call Me in the Morning” Immunity30:656-665; Brusko et al., (2008) “Human regulatory T cells: Role inautoimmune disease and therapeutic opportunities” Immunol Rev 223:371-390; Trzonkowski et al., (2009) “First-in-man clinical results ofthe treatment of patients with graft versus host disease with human exvivo expanded CD4+CD25+CD127− T regulatory cells” Clin Immunol 133:22-26; Brunstein et al., “Alternative donor transplantation afterreduced intensity conditioning: results of parallel phase 2 trials usingpartially HLA-mismatched related bone marrow or unrelated doubleumbilical cord blood grafts” Blood 118: 282-288; and Safinia et al.,(2010) “Adoptive regulatory T cell therapy: Challenges in clinicaltransplantation” Curr Opin Organ Transplant 15: 427-434.

One possible alternative to circumvent these issues is to generateadaptive or induced Treg (iTreg) from the patient's own naïve T cellseither ex vivo or in vivo. Past reports have demonstrated that IL-2 andtransforming growth factor β1 (TGF-β) can induce a Treg cell phenotypehaving functional characteristics from naïve T cells upon in vitrostimulation. Fu et al. (2004) “TGF-beta induces Foxp3+ T-regulatorycells from CD4+CD25− precursors” American Journal of Transplantation 4:1614-1627; and Chen et al., (2003) “Conversion of Peripheral CD4+CD25−Naive T Cells to CD4+CD25+ Regulatory T Cells by TGF-beta Induction ofTranscription Factor Foxp3” Journal of Experimental Medicine198:1875-1886. However, TGF-β-induced Treg cells (TGFβ-iTreg cells) havebeen shown to be unstable in long term in vitro cultures and uponantigenic re-stimulation. Floess et al., (2007) “Epigenetic control ofthe foxp3 locus in regulatory T cells” PLoS Biology 5: 0169-0178.Additionally, the presence of inflammatory cytokines such as IL-6 canantagonize TGF-β-mediated induction of Treg cells, making the presenceof such inflammatory mediators a potential impediment to inducing Tregcells in vivo at the site of a diseased tissue. Veldhoen et al., (2006)“TGF-beta in the context of an inflammatory cytokine milieu supports denovo differentiation of IL-17-producing T cells” Immunity 24: 179-189;and Bettelli et al., (2006) “Reciprocal developmental pathways for thegeneration of pathogenic effector TH17 and regulatory T cells” Nature441: 235-238.

Numerous reports suggest that these problems might be overcome throughthe use of small molecules that work in concert with TGF-β to induceTreg cells. For example, all-trans retinoic acid (RA) is known topotently synergize with IL-2 and TGF-β to induce FoxP3 expression innaïve T cells and allows for induction of Treg cells even in thepresence of inflammatory cytokines. Thorough characterization of thephenotype and function of RA-induced Treg (RA-iTreg) cells demonstratessuppressor activity and are more stable than TGFβ-iTreg cells.Nevertheless, RA-iTreg cells have a specific disadvantage in that theymigrate primarily to the mucosal tissues in the gut, which might limittheir use. Mucida et al., (2007) “Reciprocal TH17 and regulatory T celldifferentiation mediated by retinoic acid” Science 317: 256-260; and Luet al., (2011) “Characterization of Protective Human CD4+CD25+ FOXP3+Regulatory T Cells Generated with IL-2, TGF-β and Retinoic Acid” PLoSONE 5: 1-12; and Benson et al., (2007) “All-trans retinoic acid mediatesenhanced T reg cell growth, differentiation, and gut homing in the faceof high levels of co-stimulation” Journal of Experimental Medicine 204:1765-1774.

Further, other evidence suggests additional disadvantages to RA-iTregcells in that, depending on the immunological microenvironment, RA caninduce inflammation rather than tolerance. DePaolo et al., (2011)“Co-adjuvant effects of retinoic acid and IL-15 induce inflammatoryimmunity to dietary antigens” Nature 471: 220-224. Also, RA has beenshown to induce hypervitaminosis-A upon local administration, and henceit would be difficult to use this combination (cytokines+RA) to induceTreg cells in vivo. Jones D H, (1989) “The role and mechanism of actionof 13-cis-retinoic acid in the treatment of severe (nodulocystic) acne”Pharmacol Ther 40: 91-106; and Barua et al., (1996) “Percutaneousabsorption, excretion and metabolism of all-trans-retinoylbeta-glucuronide and of all-trans-retinoic acid in the rat” SkinPharmacol 9: 17-26.

Another small molecule that synergizes with IL-2 and TGF-β to induceFoxP3 expression in naïve T cells is the serine/threonine protein kinaseinhibitor rapamycin (RAPA). It has been demonstrated that, like RA, RAPAcan induce Treg cells even in the presence of IL-6. However, thephenotype and function of RAPA-induced Treg (RAPA-iTreg) cells has yetto be characterized. Kopf et al., (2007) “Rapamycin inhibitsdifferentiation of Th17 cells and promotes generation of FoxP3+ Tregulatory cells” Int Immunopharmacol 7: 1819-1824; Haxhinasto et al.,(2008) “The AKT-mTOR axis regulates de novo differentiation ofCD4+Foxp3+ cells” J Exp Med 205: 565-574; and Cobbold et al., (2009)“Infectious tolerance via the consumption of essential amino acids andmTOR signaling” Proc Natl Acad Sci USA 106: 12055-12060.

IV. Conventional Controlled Release Formulations

Several drug delivery systems are known that provide for a roughlyuniform and controllable rate of release. A variety of different mediaare described below that are useful in creating drug delivery systems.

Microparticles generally refer to compositions including, but notlimited to, nanoparticles, microspheres, nanospheres, microcapsules, andnanocapsules. Preferably, some microparticles contemplated by thepresent invention comprise poly(lactide-co-glycolide), aliphaticpolyesters including, but not limited to, poly-glycolic acid andpoly-lactic acid, hyaluronic acid, modified polysacchrides, chitosan,cellulose, dextran, polyurethanes, polyacrylic acids, psuedo-poly(aminoacids), polyhydroxybutrate-related copolymers, polyanhydrides,polymethylmethacrylate, poly(ethylene oxide), lecithin andphospholipids. Microparticles are generally distinguished fromliposomes, as microparticles are usually made of organic polymers, whileliposomes are usually made of lipids, such as phospholipids.

Microspheres and microcapsules are useful due to their ability tomaintain a generally uniform distribution, provide stable controlledcompound release and are economical to produce and dispense.Microspheres are obtainable commercially (Prolease®, Alkerme's:Cambridge, Mass.). For example, a freeze dried medium comprising atleast one therapeutic agent is homogenized in a suitable solvent andsprayed to manufacture microspheres in the range of 20 to 90 μm.Techniques are then followed that maintain sustained release integrityduring phases of purification, encapsulation and storage. Scott et al.,“Improving Protein Therapeutics With Sustained Release Formulations”Nature Biotechnology, Volume 16:153-157 (1998).

Modification of a microsphere composition by the use of biodegradablepolymers can provide an ability to control the rate of therapeutic agentrelease. Miller et al., “Degradation Rates of Oral Resorbable Implants.Polylactates and Polyglycolates: Rate Modification and Changes inPLA/PGA Copolymer Ratios” J. Biomed. Mater. Res., Vol. II:711-719(1977).

Alternatively, a sustained or controlled release microsphere preparationis prepared using an in-water drying method, where an organic solventsolution of a biodegradable polymer metal salt is first prepared.Subsequently, a dissolved or dispersed medium of a therapeutic agent isadded to the biodegradable polymer metal salt solution. The weight ratioof a therapeutic agent to the biodegradable polymer metal salt may forexample be about 1:100000 to about 1:1, preferably about 1:20000 toabout 1:500 and more preferably about 1:10000 to about 1:500. Next, theorganic solvent solution containing the biodegradable polymer metal saltand therapeutic agent is poured into an aqueous phase to prepare anoil/water emulsion. The solvent in the oil phase is then evaporated offto provide microspheres. Finally, these microspheres are then recovered,washed and lyophilized. Thereafter, the microspheres may be heated underreduced pressure to remove the residual water and organic solvent.

Other methods useful in producing microspheres that are compatible witha biodegradable polymer metal salt and therapeutic agent mixture are: i)phase separation during a gradual addition of a coacervating agent; ii)an in-water drying method or phase separation method, where anantiflocculant is added to prevent particle agglomeration and iii) by aspray-drying method.

Controlled release microcapsules may be produced by using knownencapsulation techniques such as centrifugal extrusion, pan coating andair suspension. Such microspheres and/or microcapsules can be engineeredto achieve desired release rates. For example, Oliosphere® (Macromed) isa controlled release microsphere system. These particular microsphere'sare available in uniform sizes ranging between 5-500 μm and composed ofbiocompatible and biodegradable polymers. Specific polymer compositionsof a microsphere can control the therapeutic agent release rate suchthat custom-designed microspheres are possible, including effectivemanagement of the burst effect. ProMaxx® (Epic Therapeutics, Inc.) is aprotein-matrix delivery system. The system is aqueous in nature and isadaptable to standard pharmaceutical delivery models. In particular,ProMaxx® are bioerodible protein microspheres that deliver both smalland macromolecular drugs, and may be customized regarding bothmicrosphere size and desired release characteristics.

A microsphere or microparticle may comprise a pH sensitive encapsulationmaterial that is stable at a pH less than the pH of the internalmesentery. The typical range in the internal mesentery is pH 7.6 to pH7.2. Consequently, the microcapsules should be maintained at a pH ofless than 7. However, if pH variability is expected, the pH sensitivematerial can be selected based on the different pH criteria needed forthe dissolution of the microcapsules. The encapsulated compound,therefore, will be selected for the pH environment in which dissolutionis desired and stored in a pH preselected to maintain stability.Examples of pH sensitive material useful as encapsulants are Eudragit®L-100 or S-100 (Rohm GMBH), hydroxypropyl methylcellulose phthalate,hydroxypropyl methylcellulose acetate succinate, polyvinyl acetatephthalate, cellulose acetate phthalate, and cellulose acetatetrimellitate. In one embodiment, lipids comprise the inner coating ofthe microcapsules. In these compositions, these lipids may be, but arenot limited to, partial esters of fatty acids and hexitiol anhydrides,and edible fats such as triglycerides. Lew C. W., “Controlled-Release pHSensitive Capsule And Adhesive System And Method” U.S. Pat. No.5,364,634 (herein incorporated by reference).

Microparticles may also comprise a gelatin, or other polymeric cationhaving a similar charge density to gelatin (i.e., poly-L-lysine) and isused as a complex to form a primary microparticle. A primarymicroparticle is produced as a mixture of the following composition: i)Gelatin (60 bloom, type A from porcine skin), ii) chondroitin 4-sulfate(0.005%-0.1%), iii) glutaraldehyde (25%, grade 1), and iv)1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDChydrochloride), and ultra-pure sucrose (Sigma Chemical Co., St. Louis,Mo.). The source of gelatin is not thought to be critical; it can befrom bovine, porcine, human, or other animal source. Typically, thepolymeric cation is between 19,000-30,000 daltons. Chondroitin sulfateis then added to the complex with sodium sulfate, or ethanol as acoacervation agent.

V. Custom-Tailored Controlled Release Profiles

In some embodiments, the present invention contemplates compositions andmethods that encapsulate and/or attach VIP into and/or ontocustom-tailored poly(lactic-co-glycolic acid) (PLGA) microspheres (MPs).For example, the custom-tailored microsphere result from a procedurethat predictably controls VIP release of microparticles as a function oftime. Such predictable controlled release of VIP was achieved by usingcustom-tailored microspheres having different polymer molecular weights.Alternatively, predictable controlled release of VIP was achieved byusing custom-tailored micro spheres having polyethylene glycol (PEG)and/or sodium chloride (NaCl) in an inner aqueous phase of themicroparticle.

In some embodiments, the present invention contemplates compositionsproviding a predictable controlled release of compounds to induce a Tregcell phenotype (e.g., determined by the expression of canonical Tregmarkers and migratory surface markers). In one embodiment, the inducedTreg phenotype includes, but is not limited to, VIP-iTreg.

In one embodiment, the present invention contemplates compositions andmethods for the development and testing of controlled releaseformulations for Treg cell induction compounds (e.g., VIP) having apredictable release profile. In other words, a predicted VIP releaseprofile is achieved with a custom-tailored predetermined kinetic andtemporal pattern. Rothstein et al., “A simple model framework for theprediction of controlled release from bulk eroding polymer matrices” JMater Chem (2008) 18:1873-1880; and Rothstein et al., “A unifiedmathematical model for the prediction of controlled release from surfaceand bulk eroding polymer matrices” Biomaterials (2009) 30:1657-1664. Asis shown herein, a specific composition of a microparticle can bedetermined, in advance, that results in the predicted release profile.The data presented herein show that VIP microparticles (VIPMPs) arecapable of Treg cell population induction in vitro using either mouse orhuman cells.

The absence of regulatory T (Treg) cells may be involved in a widevariety of disorders including, but not limited to, autoimmunity,dermatitis, periodontitis and/or transplant rejection. Logically, onepotential treatment option for these disorders would be to increaselocal Treg cell numbers. For example, enhancing local numbers of Tregcells might be achieved through in situ Treg cell expansion orinduction. Current methods for in vivo Treg cell expansion rely onbiologic therapies, which are not Treg cell-specific and are associatedwith many adverse side-effects.

A. Controlled Release Microparticle Characterization The data presentedherein use a VIPMP comprising a polymer (e.g., RG502H) having aviscosity of 0.16-0.24 dl/g. Such microparticles are spherical and haveaverage diameter of approximately 15-30 μm. Additionally, themicroparticles may comprise a slightly porous exterior surface. Theseparticles were specifically formulated to be porous by altering osmoticpressures between the inner emulsion and the outside aqueous phaseduring microparticle preparation. It was predicted that this compositionwould comprise a high initial VIP release burst followed by a continuousrelease.

VIP microparticles fabricated using a 12.6 kDa RG502H polymer had meandiameters of 16.7 μm and 17.7 μm for nonporous and porous particles,respectively. See, FIG. 31.

B. VIP Release From VIPMPs Having Pre-Determined Release Profiles

Various tactics can be employed to alter the release of encapsulated VIPfrom PLGA microspheres including, but not limited to: a) VIP release maybe decreased as polymer molecular weight increases as evident in thepresent data showing that release of VIP from 100 kDa polymer particleshad a very small initial burst followed by a region of near linearrelease; b) addition of NaCl or PEG to the inner aqueous phaseinfluences VIP release; c) porous microparticles fabricated with NaClshowed a diminished initial burst possibly due to electrostaticinteractions; d) electrostatic interactions between VIP with the PLGApolymer may delay VIP release in that VIP has an exceptionally highisoelectric point of 11.8, meaning that at pH of 7, the peptide has anet positive charge and since the polymer is negatively charged, the VIPcould aggregate with the polymer, which may be enhanced when NaCl ispresent.

Controlled release of VIP from VIPMPs made in accordance with Example Dawas measured over a 60 day period and evaluated for both a predictedlinear release profile and a predicted multi-bolus release profile.

VIP release profiles from VIP MPs were evaluated using ELISA plates. TheVIP MPs contained particles of pure polymer weight: 4.2 kDa, 12.6 kDawith and without PEG, and 100 kDa. Predictive model calculationsgenerated proposed ratios of microparticles with different polymermolecular weights (10.6% of the 4.2 kDa, 31.9% of the 12.6 kDa withoutPEG, and 57.5% of the 100 kDa polymer) that predicted a linear releasefor a period of approximately 50 days. Rothstein et al., “A simple modelframework for the prediction of controlled release from bulk erodingpolymer matrices” J of Mater. Chem. (2007) 18:1873-1880. Thisalgorithmic model further suggested that when 4.2 kDa, 12.6 kDa withPEG, and 55 kDa polymers were mixed in equal amounts (e.g., a ratio of1:1:1, or 33.3% of each polymer), the resultant microparticle would beexpected to have an extended multi-bolus release profile.

A VIP release profile for a linear VIPMPs showed an initial burstfollowed by an extended release over a period of 60 days. See. FIG. 27A.As would be expected from the algorithm prediction, the multi-bolus datashowed a secondary burst around day 15 and a possible start of atertiary burst at day 45. Further, the multi-bolus group released abouttwice as much VIP as compared to the linear VIPMP release profile. Thelinear VIPMPs released much slower than the multi-bolus VIPMPs and atday 5, release of VIP from the linear VIPMPs began releasing in anear-linear fashion. See. FIG. 27A. A comparison of the observed releaseand the model prediction of the linear VIPMPs is presented that ignoresthe initial burst occurring during the first five days of releaseprofile. See, FIG. 27B.

With the exception of the initial burst, the computer model successfullypredicted the polymer ratio necessary to achieve a near-linear releaseof VIP for a period of 50 days. Although it is not necessary tounderstand the mechanism of an invention, it is believed that that therelease of VIP may be delayed due to ionic interactions of VIP with thePLGA polymer. VIP has an exceptionally high isoelectric point of 11.8,meaning that at pH of 7, the peptide has a net positive charge. Sincethe polymer is negatively charged, the VIP could aggregate with thepolymer. It appeared that the VIP did not finish releasing from both thelinear and multi-bolus groups, although the model predicted completerelease by day 50.

The above described controlled release of VIP over an extended periodwas performed using particles formulated with mixtures of 4.2 kDa, 12.6kDa, and 100 kDa polymers. Release assays were performed in triplicatefor each batch of VIP microparticles generating cumulative releaseprofiles for each polymer molecular weight. FIG. 32. Particles of thelowest polymer molecular weight released most quickly as predicted. The100 kDa particles released markedly slower than both the 4.2 kDa and12.6 kDa particles in an almost linear fashion. The 12.6 kDa particleset showed an unexpected (e.g., anomalous) large initial burst on thefirst day.

Each release profile in FIG. 32 was compared to a release profilepredicted by a computer model. FIG. 33. The model was most accurate inpredicting release from the 4.2 kDa polymer particles. The largemagnitude of the initial burst of the 12.6 kDa batch was not predictedby the model. In fact, a second VIP release profile from 12.6 kDapolymer particles conducted over a shorter time period where the initialburst was much smaller in magnitude. See, FIG. 34. The 100 kDa particlesbegan releasing VIP earlier than the profile predicted by the model,which did not capture the near-linear release from the 100 kDaparticles.

The predictive computer modeling described herein was also used togenerate custom-tailored microparticles having a mixture of a specificratio of different polymer molecular weights (e.g., for example, 4.2 kDa(10.6%), 12.6 kDa (31.9%), and 100 kDa polymer (57.5%)). An extendedmulti-bolus release profile was predicted by computer modeling using amixture of 4.2 kDa polymers (33.3%), 12.6 kDa polymers+PEG (33.3%), and100 kDa polymers (33.3%). Cumulative release profiles of these linearand multi-bolus groups are shown. FIG. 35A. A comparison was then madeof the observed VIP release and the model prediction of the linear groupwhile ignoring the initial burst that occurred during the first fivedays of release. FIG. 35B. Both the linear and multi-bolus groups showedinitial bursts followed by extended release over the period of 60 days.The multi-bolus group released about two times as much VIP and showed asecondary burst around day 15 and a possible start of a tertiary burstat day 45. The linear batch released much slower and at day 5, releaseof VIP from the linear batch began releasing in a near-linear fashion.

The effects of NaCl or poly(ethylene glycol) (PEG) in the inner aqueousphase on release dynamics were also examined. Cumulative VIP releaseprofiles were generated using nonporous 12.6 kDa PLGA VIPMPs and porous12.6 kDa PLGA VIPMPs (made with 7.5 mm NaCl inner aqueous phase). FIG.36. The nonporous VIP microparticles experienced a slight initial burstand a secondary burst around Day 14. The porous VIP particlesexperienced less of an initial burst and slower release than thenonporous particles.

Release profiles from 12.6 kDa polymer microparticles with or without a4×10 mM PEG inner aqueous phase were also generated. FIG. 37. Theinitial burst and release rate was reduced dramatically when PEG wasadded to the inner aqueous phase. When PEG was added to the inneraqueous phase of 12.6 kDa polymer particles, the initial burst wasgreatly reduced. Interestingly, VIP release from the 12.6 kDa polymerparticles with PEG greatly resembled the release from 100 kDa polymerparticles. See FIG. 40. Although it is not necessary to understand themechanism of an invention, it is believed that PEG may minimizehydrophobic interactions with the polymer.

C. VIP Bioactivity Assays

In some examples, the data presented herein was collected in cultureddendritic cells to ascertain if VIP released from microparticlespromotes CCL22 production by steering DCs towards a tolerogenicphenotype. DCs treated with VIP microparticles were also used inchemotaxis studies to measure the recruitment of FoxP3+ CD4+ T-cells.

To show that VIP microparticles released bioactive VIP, dendritic cell(DC) cultures and chemotaxis studies were performed. Three separate DCcultures with 24 individual wells were completed in accordance withExample IVX. Both GM-CSF and IL-4 were used to culture the cells in thefirst two DC cell cultures, but for the third DC culture, the IL-4 wasomitted to reduce its possible interference in steering immune cellstoward a tolerogenic phenotype. Chemotaxis studies of CD4+ T-cells wereperformed and the percent of FoxP3+ T-cells which flowed through thetranswells for each group was evaluated. The data is presented as anormalized percent of FoxP3+ cells which were recruited by the DCs forthe first DC when both IL-4 and GM-CSF were used to culture the DCprecursors and when GM-CSF was used alone in the last DC culture. TheDCs treated with soluble VIP or releasates from VIP microparticlesrecruited a higher percentage of FoxP3+ cells than DCs that onlyreceived LPS or releasates from blank particles. See, FIG. 28.

DCs cultured in the absence of IL-4 showed similar trends in therecruitment of regulatory T cells, suggesting that VIP itself (not IL-4)is responsible for CCL22 induction. When the DC culture was repeatedwith the absence of IL-4 and addition of 5×GM-CSF, flow cytometryindicated slightly different trends. The DCs treated with LPS alone,soluble VIP, and releasates from VIP microparticles recruited the samepercentage of FoxP3+ cells. The DCs treated with releasates from theblank microparticle groups recruited less FoxP3+ cells than the othergroups. The distinction between the VIP microparticle group and theblank microparticle group was increased when no IL-4 was used.

In the in vitro DC cultures, a higher percentage of FoxP3+ regulatoryT-cells after chemotaxis indicated that the DCs produced more CCL22, achemokine known to be involved in regulatory T cell recruitment. Thereleased VIP appeared to be bioactive since the DCs treated withreleasates from VIP microparticles showed higher percentage recruitmentof FoxP3+ cells than those treated with blank releasates for all threeexperiments. Studies have shown that CCL22 production of DC cells doesnot increase as significantly after treatment with soluble VIP. Delgadoet al., “VIP/PACAP preferentially attract Th2 effectors throughdifferential regulation of chemokine production by dendritic cells”(2004) FASEB J18:1453-1455. As IL-4 has been suggested to skew DCdifferentiation, IL-4 was omitted from the fourth DC culture in attemptto yield greater distinction between groups in percent FoxP3+ cellrecruitment. Robinson et al., “Chapter 17. Generation of MurineBone-Marrow-Derive Dendritic Cells” (2001) Dendritic Cell Protocols191-98. While there was a greater difference between the blankmicroparticles and VIPMPs of the DC culture without IL-4, the LPScontrol, soluble VIP, and VIP microparticle groups all appeared to showsimilar trends.

CCL-22 production was also determined from samples collected at 0, 7, 24and 48 hours from a mature DC control group and a soluble mature DCgroup. CCL-22 production was similar between the mature DC control groupand the soluble mature DC groups. CCL-22 concentration rapidly decreasedas microparticle concentration was increased in all microparticlegroups. Therefore, CCL22 production by DCs treated with a lowconcentration of nonporous microparticles relative to the control andsoluble VIP group was investigated further. CCL22 concentrationsproduced by dendritic cells was determined in the presence or absence ofVIP, both in soluble form and in microparticles, at 7, 24, and 48 hours.FIG. 39. The VIP microparticles induced more CCL22 production than theblank microparticles at each time point, gaining statisticalsignificance at 24 hours with p<0.05.

Released VIP from microparticles appeared bioactive in the in vitro DCcultures. The observed CCL22 production of DCs treated withmicroparticles was significantly higher than that of the respectiveblank microparticles and showed a significant increase at 24 hours inthe nonporous VIP group. This trend was most evident at 7 and 24 hours.At 48 hours following treatment with drugs and LPS, the concentration ofCCL22 began to equilibrate between the microparticle groups. The effectsof the presence of LPS for an extended time period have not beendetermined but are thought to be detrimental for the cells. Publishedprotocols recommend that cells should be exposed to LPS for a maximum of18 hours, but the protocol of previous studies involving VIP treatmentsto DCs did not imply washing of the LPS in a 72 hour period. Delgado etal., (2004) “VIP/PACAP preferentially attract Th2 effectors throughdifferential regulation of chemokine production by dendritic cells”FASEB J 18:1453-1455. Additionally, the microparticles themselves maynegatively impact the cells and cause lower CCL22 production by DCsrelative to the control and soluble VIP groups. The particle degradationproducts, such as PLGA fragments or lactic or glycolic acid, may disruptthe cells and may cause some cells to die.

Additionally, it was expected that the DCs cultured with soluble VIPwould show a more significant increase in CCL22 production relative tothe mature DC control group, as was shown in previous studies. Delgadoet al., (2004) “VIP/PACAP preferentially attract Th2 effectors throughdifferential regulation of chemokine production by dendritic cells”FASEB J 18:1453-1455. IL-4 has been proposed to skew DC differentiationto exhibit a regulatory phenotype and might therefore diminish thedifference in CCL22 production between the groups. Rothstein et al.,(2007) “A simple model framework for the prediction of controlledrelease from bulk eroding polymer matrices” J of Mater. Chem.18:1873-1880. In subsequent chemotaxis studies that measured FoxP3+T-cell recruitment by DCs treated with VIP microparticle releasate,omission of IL-4 during cell culture increased the distinction betweenFoxP3+ T cell recruitment. This suggested that released VIP itself (notIL-4) is responsible for CCL22 induction and shows some degree ofbioactivity in vitro.

D. In Vivo Murine Model Of Periodontitis

After VIP microparticles demonstrated bioactivity in vitro, it was shownthat they also promote therapeutic effects in vivo. In some examples,the data presented herein evaluates VIP microparticles as a treatment inan in vivo murine model of periodontitis.

Micro-computerized tomographic analysis images of mouse alveolar bonewere used to determine bone loss in mice following administration ofblank MPs, CCL22MPs, and VIPMPs. The data distinguishes thecementoenamal junction (CEJ) as a focal point on which to quantitatebone loss data using either a linear measurement or a volumetricmeasurement technique. FIG. 29. Park et al., “Three DimensionalMicro-Computed Tomographic Imaging of Alveolar Bone in Experimental BoneLoss or Repair” (2007) J. Periodontol. (2007) 78:273-281.

Micro-CT analysis has not yet been reported as a technique to evaluatealveolar bone loss in mice having periodontitis treated with VIPmicroparticles. However, micro-CT analysis has been performed on micehaving periodontitis which received CCL22MP treatment. FIG. 30.

Dissecting microscope images show alveolar bone loss in infected micetreated with blank, CCL22, and VIP microparticles. FIG. 40A. VIPmicroparticles were shown to reduce alveolar bone loss as indicated bydecreased distances between the ABC and CEJ with area quantificationsconfirming these results. FIG. 40B. Mice infected with periodontaldisease and treated with VIP microparticles showed significantly lessbone loss than infected mice that received blank microparticles (withp<0.021). As shown previously, CCL22 microparticles also produce asimilar effect.

VIP microparticles significantly reduced bone loss in mice infected withperiodontal disease relative to mice treated with blank microparticles.However, there was not a significant decrease in bone loss relative tountreated mice, possibly because the sample size of the untreated wasreduced to due to accidental fractures of the maxillae during testing.The brittleness of the untreated maxillae relative to those treated withVIP microparticles, may be a testament to the therapeutic potential ofVIP.

VI. Predicting Microparticle Formulation Release Profiles

In some embodiments, the present invention contemplates improving uponmethods comprising increasing a Treg cell:Teff cell ratio. For example,is has been reported that a combination of soluble T cell inducingfactors such as IL-2, TGF-β and rapamycin establish an environment thatfavors an increase in this ratio. Haxhinasto et al., “The AKT-mTOR axisregulates de novo differentiation of CD4+Foxp3+ cells” J Exp Med (2008)205565-574; Kopf et al., “Rapamycin inhibits differentiation of Th17cells and promotes generation of FoxP3+ T regulatory cells” IntImmunopharmacol (2007) 7:1819-1824; Cobbold et al., “Infectioustolerance via the consumption of essential amino acids and mTORsignaling” Proc Natl Acad Sci USA (2009) 106:12055-12060; and Thomson etal., “Immunoregulatory functions of mTOR inhibition” Nat Rev Immunol.(2009) 9:324-337. In one embodiment, the present invention contemplatesa method to create an improved immunosuppressive, Treg cell-inducingenvironment by providing VIPMP formulations that provide a predictableand sustained release of T cell inducing factors at a local site. In oneembodiment, the VIPMP formulation comprises a polymer (e.g., PLGA).Although it is not necessary to understand the mechanism of aninvention, it is believed that these formulations are prepared inaccordance with a pre-fabrication mathematical analysis that creates aunique microparticle composition that results in a pre-determinedrelease profile tailored for the induction and proliferation of specificTreg cell populations.

In one embodiment, the present invention contemplates a compositioncomprising a VIPMP formulation. Although it is not necessary tounderstand the mechanism of an invention, it is believed that such VIPMPformulations are as effective as soluble factors that induce Treg fromnaïve T cells. In one embodiment, the VIPMP iTreg cells provide a robustTreg cell proliferation, express canonical Treg surface markers, andsuppress naïve T cell proliferation. Further, it was observed that Treginduction and proliferation occurred even when the cells were in contactwith microparticles, suggesting that the microparticles do not haveadverse on these cells. In one embodiment, VIPMP formulations inducehuman T cells into an iTreg cell population.

In one embodiment, the present invention contemplates method comprisingadministering VIPMP populations in vivo for treating medical conditionssuch as transplant rejection and/or autoimmunity mediated by a localTreg cell population induction.

VII. Mathematical Prediction of Microparticle Release Profiles

In some embodiments, the present invention contemplates using a broadlyapplicable model for predicting controlled release that eliminates theneed for exploratory, in vitro experiments during the design of newbiodegradable matrix-based therapeutics. For example, a simplemathematical model can predict the release of many different types ofagents from bulk eroding polymer matrices without regression. Suchmodels comprise deterministically calculating the magnitude of theinitial burst and the duration of the lag phase (time before Fickianrelease) by making predictions based upon easily measured or commonlyknown parameters. This model describes the release of water-solubleagents that are discretely encapsulated in bulk eroding, polymermatrices and that dissolve rapidly, relative to the time scale ofrelease. In addition to using specific equations, the model includes twocorrelations that enable predictions with knowledge of just fiveparameters, all commonly known or easily measured prior to release.These parameters are microsphere radius (R_(p)), occlusion radius(R_(occ)), polymer degradation rate (kC_(w)), polymer initial molecularweight (M_(wo)), and agent molecular weight (M_(wA)). A regression to adesired dosing schedule generates a set of matrix design parameters toguide the fabrication of a matching controlled release therapeutic.

Briefly, the operation of the predictive algorithm can be described asfollows. Consider an initially uniform matrix of known geometrycomprised of a biodegradable polymer, such as a polyester orpolyanhydride, and with randomly distributed entrapped release agent(e.g. drug of concentration C_(Ao)), loaded below its percolationthreshold (such that agent remains discrete) to ensure matrix mediatedrelease. This agent can either be dispersed as crystals (such as in thecase of uniformly loaded systems, e.g. single emulsion-basedparticulates) or housed as a solution in occlusions (e.g. doubleemulsion-based particulates). At time zero, an aqueous reservoir beginsto hydrate the matrix, a process which happens quickly for the bulkeroding polymers matrices considered herein. As the matrix hydrates,encapsulated agent adjacent to the matrix surface (with a direct pathwayfor egress) diffuses into the reservoir in a phase typically dubbed “theinitial burst”). The relative size of the occlusion (R_(occ)) occupiedby the encapsulated agent is proportional to the magnitude of theinitial burst. As the initial burst release commences, degradation ofthe polymer begins, increasing chain mobility and effectively leading tothe formation of pores in the polymer matrix. Although a number ofmechanisms have been proposed for this heterogeneous degradationprofile, one hypothesis, which has been reinforced by experimental data,is based upon regions of varying amorphicity and crystallinity. It isbelieved that (as shown using scanning electron microscopy). These poresappear to be essential for subsequent release.

With the cumulative growth and coalescence of these pores, agents areable to diffuse towards the surface of a polymer matrix that wouldotherwise be too dense to allow their passage. Thus, a pore is definedas a region of polymer matrix with an average molecular weight lowenough to allow the release of encapsulated agent. (This is in contrastto the occlusion, which is defined as a region occupied by dissolved orsolid agent, marked by the absence of polymer matrix). Further, themolecular weight associated with release may vary for each encapsulatedagent type (small molecule, peptide, protein, etc.), leading to asize-dependent restriction for agent egress.

With a size-dependent restriction on egress established, the degradationcontrolled release of any encapsulated agent can only occur when thefollowing four conditions are satisfied. 1) The release agent must bepresent in the polymer matrix. 2) A pore must encompass the releaseagent. 3) That release agent must be able to diffuse through theencompassing pore. 4) The pore must grow and coalesce with others tocreate a pathway for diffusion to the surface.

Agent concentration within a matrix (such a microsphere, rod, or thinfilm) can be calculated from Fick's second law (Equation 1) for anypoint in time (t) or space (r), provided that the agent is not generatedor consumed in any reactions while within the matrix.

$\begin{matrix}{\frac{\partial C_{A}}{\partial t} = {\nabla\left( {D_{eff}{\nabla\; C_{A}}} \right)}} & (1)\end{matrix}$

where D_(eff) is an effective diffusivity term. Integrating C_(A)/C_(ao)over the entire matrix volume yields the cumulative fraction of agentretained in the matrix (P(t)) (Equation 2).

P(t)=V ⁻¹ ∫C _(A) /C _(Ao) dV  (2)

In turn, the cumulative fraction of agent released (R(t)), a metriccommonly used to document formulation performance, is simply (Equation3):

R(t)=1−P(t)  (3)

At the center point, line, or plane of the matrix (r=0) symmetryconditions are defined such that dC_(A)/dr=0. At the matrix surface(r=R_(p)) perfect sink conditions are specified. A boundary also existsat a depth of R_(occ) in from the matrix surface (r=R_(p)−R_(occ)) wherecontinuity conditions are defined. In the subdomain from R_(p) toR_(p)−R_(occ) (terminating one occlusion radius in from the particlesurface), agent is subject to the initial release, such that D_(eff) issimply a constant (D), reflecting the movement of agent through thehydrated occlusions abutting the matrix surface. In the subdomain from 0to R_(p)−R_(occ), agent is subject to pore-dependent release, such thatD_(eff)=Dε where D is the diffusivity of the agent through the porousmatrix and ε is the matrix porosity.

For a system of like matrices, such as microspheres or sections in athin film, that degrade randomly and heterogeneously, the accessiblematrix porosity is simply a function of time as a discrete pore has, onaverage, an equal probability of forming at any position in the polymermatrix. Hence, the time until pore formation can be calculated from thedegradation of the polymer matrix, as any differential volume containinga pore would have a lower average molecular weight than its initialvalue. Assuming that the polymer degradation rate is normallydistributed, the induction time for pore formation will also follow anormal distribution. As this pore formation is cumulative, the timedependent matrix porosity (3(t)) can be described with a cumulativenormal distribution function (Equation 4).

$\begin{matrix}{{ɛ(t)} = {\frac{1}{2}\left\lbrack {{{erf}\left( \frac{t - \overset{\_}{\tau}}{\sqrt{2\sigma^{2}}} \right)} + 1} \right\rbrack}} & (4)\end{matrix}$

In this equation, τ is the mean time for pore formation and σ² is thevariance in time required to form pores.

Calculating the cumulative normal induction time distribution (ε(t))requires values for τ and σ². For polymers that obey a first orderdegradation rate expression, the mean time for pore formation (τ) can bedetermined as follows:

$\begin{matrix}{\overset{\_}{\tau} = {\frac{- 1}{{kC}_{w}}\ln {\frac{M_{wr}}{M_{wo}}}}} & (5)\end{matrix}$

where kC_(w) is the average pseudo-first order degradation rate constantfor the given polymer type, M_(wo) is the initial molecular weight ofthe polymer, and we define M_(wr) as the average polymer molecularweight in a differential volume of matrix that permits the diffusion ofthe encapsulated agent. For blended polymer matrices, the value for τwas calculated by averaging the results obtained from Equation 5 foreach component.

It is reasonable to believe that the matrix molecular weight at release(M_(wr)), which dictates how much degradation is required before releasecan occur, would vary depending on the size of the encapsulated agent.Macromolecules or larger agents can only diffuse through a section ofmatrix if it is almost entirely free of insoluble polymer chains. Hencethe M_(wr) for such agents is considered the polymer solubilitymolecular weight (e.g., 668 Da for 50:50 PLGA). As agent size decreases(as indicated by M_(wA)), however, egress can occur through more intactsections of polymer matrix (higher M_(wr)), as less free space is neededto allow their passage.

The distribution of polymer degradation rates (kC_(w)(n)) attributed tomatrix crystallinity is needed to calculate the variance (σ²) in theinduction time distribution for pore formation (ε(t)). To determinekC_(w)(n), the first order degradation rate equationM_(w)=M_(wo)e^(−kCw)t was linearly fitted at three different timeperiods to published degradation data for the desired hydrolysablepolymer. Fitting the initial slope of the degradation profile providesthe degradation rate constant of amorphous polymer as degradation occursfaster in amorphous regions of the matrix. Fitting data from the finalweeks of degradation produces a rate constant for the crystallinematerial, as amorphous regions are largely eroded by this point.Finally, a fit of the entire degradation profile yielded a rate constantindicative of the overall morphology.

With values for kC_(w)(n) defined, a distribution of induction times(t(n)) was calculated using equation 5. For blended polymer matricesthis T(n) includes values calculated at all component kC_(w)(n) andM_(wo). The standard deviation was taken for τ(n), then divided by acrystallinity-based factor and squared, yielding an adjusted variance(σ²), which conforms with lamellar size data.

This crystallinity-based factor adjusts the probability of finding poresformed from the fastest degradation rate in kC_(w)(n) to match theprobability of finding a differential volume of matrix containing purelyamorphous polymer. For all modeled cases, this differential volume isdefined as a region large enough to allow the passage of a small virusor protein complex (20 nm diameter). As multiple lamellar stacks can fitinto this differential volume, the probability that such a volume ispurely amorphous can be calculated from of the number of stacks perdifferential volume and the average crystallinity of the matrix. Fromcrystallinity data on 50:50 PLGA matrices, the probability of finding apurely amorphous differential volume is calculated as 0.05%. Thus, toensure that the probability of finding a pore formed from the fastestdegradation rate in kC_(w)(n) also equals 0.05%, the standard deviationin the induction time distribution for pore formation was adjusted by afactor of 5. Similarly, factors of 4 and 2 were calculated fromcrystallinity data for 75:25 PLGA and polyanhydride matrices,respectively.

With values for τ and σ² selected (defining ε(t)), a finite elementsolution to Equation 1 may be calculated using a commercially availablesoftware program (e.g., Comsol®, v3.3) for the given matrix geometry,using default solver settings. To decrease computation time, the matrixgeometry can be simplified to one dimension based on symmetry, for asphere, or high aspect ratio, for a thin film. The resultingconcentration profiles are numerically integrated to calculate thecumulative fraction of agent released (Equations 2 and 3). Forvalidation, the numerical solutions of the model can be fit toexperimental data sets by varying M_(wr) and D. It should be noted thatdata points charting the kinetics of the initial burst can be omittedfrom these regressions, as the model only predicts the magnitude of thisphase. Each fit may be optimized using a commercially available softwareprogram (e.g., Matlab®, R2007a) based on a minimized sum-squared error(SSE) or weighted sum-squared error (wSSE) when error bars are provided.

The above described computer model was most accurate in predictingrelease of VIP from 4.2 kDa polymer. The large magnitude of the initialburst observed from the 12.6 kDa polymer microparticles was notpredicted by the program. However, in other release assays of VIP frommicroparticles made of the 12.6 kDa polymer, the initial burst was notas dramatic. For these other studies, the model was more accurate inpredicting the release from the 12.6 kDa polymer microparticles,although the observed release appeared to last longer than predicted.The model failed to predict the near linear release from the 100 kDapolymer microparticles and again underestimated the duration of release,suggesting that drug-polymer interactions between VIP and the PLGApolymer may be delaying release. While the model struggled to capturethe dynamics of the microparticles consisting of either 12.6 kDapolymers or 100 kDa polymers, it was fairly accurate in predicting thata mixture of a specific ratio of polymers would generate a specificlinear release profile if the initial burst was disregarded. The initialobserved release from the mixture of 4.2 kDa, 12.6 kDa, and 100 kDapolymers resembled release from the pure 4.2 kDa polymer but was notcaptured by the model. The extended near linear release was most likelyproduced by the high percentage of 100 kDa polymer particles in themixture.

VIII. VIPMP Kits

In another embodiment, the present invention contemplates kits for thepractice of the methods of this invention. The kits preferably includeone or more containers containing a VIPMP composition to perform methodsof this invention. The kit can optionally include a pharmaceuticallyacceptable excipient and/or a delivery vehicle. The reagents may beprovided suspended in the excipient and/or delivery vehicle or may beprovided as a separate component which can be later combined with theexcipient and/or delivery vehicle. The kit may optionally containadditional therapeutics to be co-administered with the microparticlecompositions.

The kits may also optionally include appropriate systems (e.g. opaquecontainers) or stabilizers (e.g. antioxidants) to prevent degradation ofthe reagents by light or other adverse conditions.

The kits may optionally include instructional materials containingdirections (i.e., protocols) providing for the use of the reagents inthe performance of the methods described herein. In particular thedisease can include any one or more of the disorders described herein.While the instructional materials typically comprise written or printedmaterials they are not limited to such. Any medium capable of storingsuch instructions and communicating them to an end user is contemplatedby this invention. Such media include, but are not limited to electronicstorage media (e.g., magnetic discs, tapes, cartridges, chips), opticalmedia (e.g., CD ROM), and the like. Such media may include addresses tointernet sites that provide such instructional materials.

In one embodiment, the present invention contemplates a kit comprisingVIPMP populations to provide an ‘off-the-shelf’ therapeutic for creatinga local immunosuppressive environment and increasing the presence ofTreg cells at sites of inflammation. In one embodiment, the kits may beused to treat various medical conditions including, but not limited to,contact dermatitis, periodontitis and diseases where immune homeostasisis lost and needs to be restored, skin disorders, composite tissuetransplantation, prevention of graft rejection. Although it is notnecessary to understand the mechanism of an invention, it is believedthat VIPMP formulations can be administered to treat diseases anddisorders while the systemic immune system integrity such that theimmune system can continue to fight infections and inhibit malignancies.

IX. Immunological Tolerance

In developing a strategy to replace and/or decrease post-transplantimmunosuppressant therapy, “reprogramming” the immune system toselectively accept a foreign antigen without an systemic immunedown-regulation is seen as a desirable option. This state may bereferred to as a permanent immunological tolerance. One modulator ofimmunological tolerance is suspected to be regulatory T-cells (T_(reg)).T_(reg) cells have been shown to be capable of antigen-independentimmune suppression and can be activated by immature or tumor-manipulateddendritic cells, thereby inducing immunosuppression. In vitro, T_(reg)cells respond to several known soluble factors and even latex beadscoated with monoclonal antibodies specific for several T_(reg) cellsurface receptors. Tumors actively secrete compounds to modulateregulatory T-cells so as to evade immune recognition. Although notintuitively obvious, it is believed that the induction of permanentallograft tolerance may be achieved by mimicking tumor-inducedimmunosuppression.

CD4+CD25+ regulatory T-cells are believed to express the CCR4 receptorwhich binds to the chemokine CCL22. CCL22 was reported to be involved inthe migration of regulatory T-cells to tumor sites because tumors alsoactively secrete CCL22. Curiel et al., Nat Med 10:942-949 (2004). Thisrepresents just one of the methods that tumors have for evading immunerecognition. Evasion of an immune response provides for tumor survivalgiven that many of its basic biological features such as geneticinstability, invasive growth, and tissue disruption are inherentlypro-inflammatory. Besides attracting regulatory T-cells to its vicinity,tumors are believed to also have the ability to influence biologicalantigen presenting cells (i.e., for example, biological dendritic cells)to down-regulate processing and presentation of tumor associatedantigens, inhibit co-stimulatory expression (i.e. retain immaturephenotype), and alter their cytokine secretion profile towards Immunetolerance. Pardoll, D., Annual Review of Immunology 21:807-839 (2003).It has been reported that tumors also secrete TGF-β, which may influenceregulatory T-cell mediated tolerance. Chen et al., Proc Natl Acad SciUSA 102:419-424 (2005). Although it is not necessary to understand themechanism of an invention, it is believed that these other mechanismssimilar to those of tumor immune evasion and survival may also be usefulin inducing allograft tolerance.

Control over the induction of immunological tolerance would affordpermanent allograft acceptance, bypassing a lifelong regimen ofimmunosuppressive agents. Although it is not necessary to understand themechanism of an invention, it is believed that this tolerance can bemediated by activating suppressive regulatory lymphocytes (i.e., forexample, regulatory T cells).

In one embodiment, the present invention contemplates atherapeutically-relevant, modular platform to deliver multiple solublefactors and membrane surface-bound factors. In one embodiment, thesefactors are delivered in vivo by artificial particles into the vicinityof regulatory T cells (T_(reg) cells). In one embodiment, the deliveredfactors modulate T_(reg) cell proliferation. In one embodiment, thedelivered factors modulate T_(reg) cell immunosuppressive capacity. Inone embodiment, the delivered factors induce Treg cell chemotaxis. Inone embodiment, the platform is delivered in vivo. In one embodiment,the platform comprises a biocompatible and/or biodegradable syntheticconstruct, such as an artificial antigen presenting cell (aAPC). In oneembodiment, the aAPC is approximately the size of a biological cell. Inone embodiment, the aAPC comprises covalently-bound monoclonalantibodies and/or encapsulated soluble cytokines/chemokines. In oneembodiment, the antibodies and cytokine/chemokines stimulate lymphocyteproliferation. In one embodiment, the lymphocyte stimulation isperformed in vitro. In one embodiment, the lymphocyte stimulation isperformed in vivo.

In one embodiment, the present invention contemplates a constructcomprising a pattern of membrane surface factors such that the patternmimics a biological dendritic cell/regulatory T-cell immunologicalsynapse (IS). In one embodiment, the pattern increases the stimulatorycapacity of the construct. In one embodiment, the synapse comprises a“bull's-eye” pattern. In one embodiment, the “bull's-eye” patterncomprises an apical region and an equatorial region. In one embodiment,the pattern further comprises a region between the apical and equatorialregions.

In one embodiment, the present invention contemplates a method forcreating an artificial biomimetic immunological synapse. In oneembodiment, the synapse is discrete and topologically complex. In oneembodiment, the synapse comprises at least two immunosuppressivefactors. In one embodiment, the synapse is located on the surface of anartificial presenting cell. In one embodiment, the synapse comprises a“bull's-eye” pattern. In one embodiment, the “bull's-eye” patterncomprises an apical region and an equatorial region. In one embodiment,the pattern further comprises a region between the apical and equatorialregions.

A. Tolerogenic Cells

The biological processes whereby tolerance is induced involve thesuppression or deletion of alloreactive T-cells which would otherwisedestroy an allograft. This process is thought to be mediated by a subsetof regulatory T-cells which represent 5-10% of all peripheral CD4+lymphocytes. Mahnke et al., Blood 101:4862-4869 (2003).

Suppressive lymphocyte involvement in immune response was firstsuggested in the early 1970s. Gershon et al., Immunology 21:903-914(1971). Recently, however, a suppressive regulatory T-cell (T_(reg)) hasbeen implicated in the development of immune tolerance. For example, apopulation of these lymphocytes that express high levels of CD25, the asubunit of the IL2 receptor, have been reported as an immune suppressor.Sakaguchi et al., J Immunol 155:1151-1164 (1995).

T_(reg) cells also express a unique transcription factor called FoxP3,which is thought to be required for the development of T_(reg)suppressive capacity and is a recognized marker for T_(reg) cells. Horiet al., Science 299:1057-1061 (2003); and Fontenot et al., Nat Immunol4:330-336 (2003). Furthermore, it is believed that the extent ofexpression of this transcription factor can correlate with thesuppressive capacity and activation state of a T_(reg) cell. It has beenshown that transfer of the gene for FoxP3 into cells which are CD4+CD25−confers regulatory capacity which is otherwise absent. Fontenot et al.,Nat Immunol 4:330-336 (2003). T_(reg) cells may also express high levelsof CTLA-4 (i.e., for example, CD152) which may also be involved in theirregulatory capacity as this molecule can bind to the B7 class ofco-stimulatory molecules in place of CD28 thereby resulting in theproduction of transforming growth factor-β, or TGF-β. Perez et al.,Immunity 6:411-417 (1997). TGF-β, along with engagement of the T-cellreceptor, has been demonstrated to differentiate naïve, peripheral CD4+CD25− T-cells into CD4+CD25+ cells with suppressive capacity, suggestingthat this factor may be important for the in vivo generation andmaintenance of T_(reg) cells. Walker et al., J Clin Invest 112:1437-1443(2003). Regulatory T-cells are also believed to express chemokinereceptors including, but not limited to, CCR4 and CCR8, rendering themfully capable of migration (i.e., for example, by chemotaxis) to a siteof inflammation or to the lymph nodes upon appropriate signaling. Iellemet al., J Exp Med 194:847-853 (2001).

Mechanisms of action used by regulatory T-cells in promoting tolerancein vivo has been a topic of dispute. Some believe that cell-to-cellcontact between T_(reg) cells and the alloreactive lymphocytes providethe activation stimulus that results in immunosuppression. Othersbelieve that specific stimulatory factors (i.e., for example, solublefactors and/or membrane surface bound factors) which have been shown tobe secreted from regulatory T-cells may be responsible forcontact-independent suppression (i.e., for example, TGF-β, IL-10,CTLA-4, and IL-4). For example, studies which support the latterhypothesis demonstrate that: i) T_(eg) activation by TNF-β may beblocked by monoclonal antibodies (Belghith et al., Nat Med 9:1202-1208(2003); and ii) T_(reg) activation is diminished in TGF-β receptorknock-out mice (Green et al., Proc Natl Acad Sci USA 100:10878-10883(2003)). However, it has been reported that suppression by T_(reg) cellsin vitro is not inhibited by blocking TGF-β and suggest a cellularcontact dependent mechanism. Piccirillo et al., J Exp Med 196:237-246(2002).

Nevertheless, one interesting notion regarding the method of T_(reg)cell suppression which is gaining popular acceptance is an antigenindependent suppression mechanism, known as “bystander regulation”.Several new reports are providing evidence for “bystander regulation”which opposes a previous hypothesis that regulatory T-cellspreferentially mediate immunosuppression in an antigen specific mannerGraca et al., Proc Natl Acad Sci USA 101:10122-10126 (2004); and Karimet al., Blood 105:4871-4877 (2005). In particular, Karim et al. reportedthat CD25+CD4+ T_(reg) cells generated by nominal antigens under coverof anti-CD4 monoclonal antibody treatments were fully capable oftolerizing mice to cardiac allografts comprised of completely differentantigens. Therefore, although most studies show that T-cell receptorstimulation seems to be involved in tolerization, antigen specificitymay or may not be limiting in the overall effects of immunosuppressioninduced by regulatory T-cells. The data presented herein suggest thatT_(reg) cell immunosuppression may be meditated through a combination ofcontact dependent and independent mechanisms.

1. Antigen Presenting Cells

In one embodiment, the present invention contemplates methods that wouldreconcile the apparently conflicting in vitro and in vivo data regardingcytokine-dependent suppressive capacity. In one embodiment, the methodcomprises using cell types that are not present in the in vitroexperimental models (i.e., for example, a co-mediator cell). Although itis not necessary to understand the mechanism of an invention, it isbelieved that these co-mediator cells may secrete their ownimmunomodulating cytokines or act in other suppressive capacities at thesame time as regulatory T-cells. For example, a co-mediator cell maycomprise an antigen presenting cells (APCs). Antigen presenting cellsare also referred to as dendritic cells and have been shown to have atolerogenic capacity. Matzinger et al., Nature 338:74-76 (1989);Steinman et al., Proc Natl Acad Sci USA 99:351-358 (2002); Brocker etal., J Exp Med 185:541-550 (1997); and Volkmann et al., J Immunol158:693-706 (1997).

APCs and lymphocytes may interact at a contact point in accordance witha “two signal” model of stimulation. For example, the presence of T-cellreceptor (TCR)/Major Histocompatibility Complex (MHC) engagement (i.e.,for example, Signal 1) without the presence of a proper co-stimulatorymolecule engagement (i.e., for example, Signal 2) may lead to lymphocyteprogression toward a state of anergy or deletion through apoptosis. Insupport of this hypothesis, it has been demonstrated that engagement ofthe T-cell receptor using monoclonal antibodies specific for CD3 (i.e.,for example, T-cell receptor subunit believed responsible for signaltransduction), in the absence of co-stimulation, led to long lastingproliferative unresponsiveness in murine T-cell clones. Jenkins et al.,J Immunol 144: 16-22 (1990).

Dendritic cells (i.e., for example, APCs) in an immature state (i.e.that have not been stimulated by inflammatory factors) have beenobserved to possess a low level expression of co-stimulatory moleculeswhile still maintaining expression of MHC Class I and II. These immaturedendritic cells have a high capacity to take up material byreceptor-mediated endocytosis, pinocytosis, and phagocytosis and arethought to survey the periphery and constantly process and presentself-antigen. These observations suggest that an immunological tolerantstate is maintained in the absence of co-stimulatory molecules even whenself-antigen is persistently presented. Consequently, it would bereasonable to believe that the presence of co-stimulatory moleculesrepresent danger signals. Matzinger, P. Science 296:301-305 (2002).Although it is not necessary to understand the mechanism of aninvention, it is believed that this would explain why autoimmunitydevelops when the normal, perpetual tolerant state is somehow disrupted.

In light of the above, it is most likely not a coincidence thatpersistent expression of antigen in the periphery is also thought to besufficient for generation of regulatory T-cells. Taams et al., Eur JImmunol 32:1621-1630 (2002). Furthermore, soluble factors are secretedby T_(reg) cells that generate and maintain APCs in an immature stateincluding, but not limited to: i) IL10 (De Smedt et al., Eur J Immunol27:1229-1235 (1997)); ii) TGF-β (Geissmann et al., J Immunol162:4567-4575 (1999)); and IL4 (Inaba et al., Journal of ExperimentalMedicine 176:1693-1702 (1992)). On the other hand, others have shownthat immature/tolerogenic dendritic cells also have the ability tomediate contact-dependent stimulation of T_(reg) cells. These secretedsoluble factors are also suggested to generate and maintain T_(reg)cells thereby creating the possibility for feedback regulation. Steinmanet al., Annu Rev Immunol 21:685-711 (2003). This feedback mechanismmight even extend to the apoptosis of dendritic cells which are alreadyactivated or mature by regulatory T-cells. Frasca et al., J Immunol168:1060-1068 (2002). These data suggest that APCs and T_(reg) cellsinteract to modulate immunotolerance.

The current understanding of biological processes whereby regulatoryT-cells (T_(reg)) promote tolerance is presented in Table 1.

TABLE 1 Summary Of T_(reg) Observations Observation SignificanceReference Tolerogenic T_(reg) cells express These markers can aid in theSakaguchi et al., J Immunol high surface levels of CD4 & isolation ofT_(reg) cells 155: 1151 (1995). CD25. The transcription factor Foxp3Activation state of T_(reg) can be Fontenot et al., Nat Immunol ishighly expressed in determined by measuring 4: 330 (2003). activatedT_(reg). Foxp3. TGF-β1 can generate T_(reg) TGF-β1 is important toBelghith et al., Nat Med while inhibiting other T-cells. preferentialdevelopment of T_(reg). 9: 1202 (2003); and Green et al., PNAS 100:10878 (2003). T_(reg) express the chemokine Chemokines such as CCL22Chen et al., American receptors CCR4 and CCR8 may be used topreferentially Journal of Transplantation and are attracted by theirrecruit T_(reg). 6: 1518 (2006); and ligands. Curiel et al., Nat Med 10:942 (2004). Tolerogenic dendritic cells Contact-mediated and solubleSteinman et al., Annu Rev have been associated with, and factorstimulation provided by Immunol 21: 685 (2003). have the capacity tostimulate, these APCs can generate T_(reg) T_(reg). T_(reg) stimulatedby a particular T_(reg) are capable of promoting Karim et al., Bloodantigen can mediate immune allograft tolerance regardless 105: 4871(2005). suppression in the presence of of the antigen/TCR-stimulus anallograft without this used to generate them. antigen. Beads coated withmAb A similar strategy could be Hoffmann et al., Blood specific to CD3and CD28 in utilized in vivo by including 104: 895 (2004). the presenceof IL2 were soluble factor(s) in a capable of stimulating andbiodegradable format. proliferating T_(reg) in vitro.

Although it is not necessary to understand the mechanism of aninvention, it is believed that the stimulation, and behavior of, T_(reg)cells can generate immune tolerance. Professional antigen presentingcells (APCs) (i.e., for example, dendritic cells: DCs) have also beenshown to have tolerogenic capacity, although the precise mechanism oftheir suppressive function is unclear. Matzinger et al., Nature 338:74(1989); and Steinman et al., Proc Natl Acad Sci USA, 99:351 (2002). Arelationship between DCs and T_(reg) cells has been observed where apersistent presentation of antigen by tolerogenic DCs generates T_(reg)cells through both contact-mediated and secreted soluble signal-mediatedstimulation. Steinman et al., Annu Rev Immunol 21:685 (2003). Thus, APCsmight mediate immunosuppression by stimulating T_(reg) cells through adual pronged mechanism.

Although it is not necessary to understand the mechanism of aninvention, it is believed that an artificial particles comprising Tregcell factors should modulate T_(reg) cells in a manner similar tobiological antigen presenting cells. For example, a membrane surfacepresentation (i.e., for example, an immunological synapse) ofstimulating protein factors on a biological dendritic cell is notdisplayed in a random pattern. In one embodiment, the present inventioncontemplates an artificial antigen presenting cell comprising solubleand/or membrane bound stimulating factors displayed in non-randompatterns.

In particular, biological antigen presenting cells have been reported todevelop a mature immunological synapse (IS) and are composed ofdiscretely defined spatial regions of receptors. Monks et al., Nature395:82 (1998). Some in the art have termed these regions assupramolecular activation clusters, or SMACs. Grakoui et al., Science285:221 (1999). A central SMAC (cSMAC) region may comprise areasproviding factors for TCR stimulation and/or soluble factors forco-stimulation. The cSMAC may be surrounded by at least one peripheralSMAC (pSMAC) region that are usually rich in adhesion molecules (i.e.,for example, ICAM). Further, pSMACs may be surrounded by a distal SMAC(dSMAC) region that may have, for example, a high CD45 density. Using adual probe fluorescence imaging technique, the SMAC spatial organizationresembles a “bulls-eye”. See, FIG. 3.

2. In Vitro T_(reg) Stimulation

In vitro testing with primary, regulatory T-cells provides a way todetermine if the soluble factors encapsulated within an artificialparticle are biologically active. Preferred assays are directly relatedto the biological activity of the encapsulated factors with respect totheir modulatory effects on T_(reg) cells. In vitro primary T_(reg)cells also provide a testing platform for the optimization of artificialparticles in preparation for therapeutic in vivo administration.

A method for large scale in vitro expansion of T_(reg) cells has beenreported. Hoffmann et al., Blood 104:895 (2004). In this study, theproliferation and suppressive capacity of cultured T_(reg) cells wasincreased using latex beads randomly coated with soluble IL2 andCD3/CD289 monoclonal antibodies. It was unreported if the T_(reg) cellpopulations produced by Hoffmann et al. are functionally the same asthose produced in vivo by biological antigen presenting cells.Sufficient discrepancies are observed between in vitro and in vivosuppressive mechanisms to indicate that they are not functionally thesame. Belghith et al., Nat Med 9:1202 (2003); and Green et al., PNAS100:10878 (2003). Such a problem in the art identifies a need to studyin vivo T_(reg) cell activation. A significant disadvantage of theHoffmann et al. technique, is that the random distribution stimulatoryfactors on the latex beads did not present the T_(reg) cells with anSMAC IS complex.

Nonetheless, in vitro data is provided herein demonstrating thestimulatory capacity of synthetic dendritic cells by several subsets ofT-cells. For example, primary lymphocytes were harvested and incubatedwith synthetic dendritic cells and the resulting level of activationwere measured.

Commercially available cell isolation kits (i.e., for example, MACS) maybe used to harvest CD4+CD25+ T_(reg) and CD4+CD25− helper T-cells fromlymph nodes of B6 mice. Once incubated with artificial antigenpresenting cells, regulatory T-cells can be examined for proliferativecapacity using techniques including, but not limited to, tritiatedthymidine incorporation, CSFE dilution, or Flow Cytometry. Cytokinesecretion profiles (i.e., for example, IL-2 or IFN-gamma) may bedetermined using ELISPOT. To assess regulatory T-cell activation thepresence of the intracellular transcription factor, FoxP3, may also bemeasured in permeabilized cells using Flow Cytometry.

To verify the exclusivity of T_(reg) cell stimulation, artificialantigen presenting cells can also be incubated with other lymphocytepopulations (CD4+CD25−, and CD8+) which are then tested for knownactivation markers. Although it is not necessary to understand themechanism of an invention, it is believed that because of the release ofTGF-β1 by aAPCs these non-suppressor cells will not be stimulated andmay actually even gain suppressive capacity. Fu et al., Am J Transplant4:1614 (2004). It is further believed that the removal of CD28co-stimulation may result in anergy of helper T-cells. Ragazzo et al.,PNAS 98:241 (2001). Thus, it is not presumed that a cSMAC region mustcontain both membrane bound TCR stimulation factors and solubleco-stimulation factors simultaneously to achieve preferential regulatoryT-cell activation.

Nonetheless, T_(reg) cells may be studied in vitro using artificial APCscomprising multiple membrane surface-bound stimulatory factors andsoluble secretable stimulatory factors. For example, the activity ofencapsulated soluble stimulatory factors may be tested using primaryT_(reg) cells in vitro by assays measuring effects on celldifferentiation, cell proliferation, and/or chemotaxis. In someembodiments, a plurality of aAPC formulations may be used wherein eachaAPC formulation comprises a different amount of both surface boundfactors and soluble releasable factors. In one embodiment, the presentinvention contemplates a method comprising identifying an optimizedformulation.

3. In Vivo Effects of Artificial Particles on Regulatory T Cells(T_(regs))

In one embodiment, the present invention contemplates a methodcomprising implanting artificial particles in vivo wherein T_(reg) cellsare recruited and/or activated. In one embodiment, the T_(reg) cellrecruitment and/or activation induces biological graft tolerance.Although it is not necessary to understand the mechanism of aninvention, it is believed that the biological graft tolerance is aresult of T_(reg) cell induced immunosuppression.

Before T_(reg) cells can be directly manipulated by an artificialparticle, however, they need to be attracted to an artificial particle.In one embodiment, the present invention contemplates an artificialparticle comprising an encapsulated chemotactic factor (i.e., forexample, CCL22). Alternatively, the T_(reg) cell population may beincreased by performing a T_(reg) cell adoptive transfer. This procedureis especially useful during investigational and/or techniqueverification studies wherein the transferred T_(reg) cells are labeledwith green fluorescent protein (i.e., for example, collected fromtransgenic GFP+ B6 mice). The resulting enriched T_(reg) population invivo facilitates histological and flow cytometry analyses. Further, anoptimization of the chemotactic factor is most likely necessary becauseof inherent differences between in vivo and in vitro techniques, asdiscussed above.

The embodiments of the present invention contemplate manytransplantation models for use as testing models. In some embodiment,testing models include, but are not limited to, skin allograft orheterotopic heart transplants, and are used to compare high and lowamounts of donor derived antigen presenting cells. These donorbiological particles may interfere or enhance the function of theartificial particles. However, it should be theoretically feasible todrastically outnumber the biological antigen presenting cells throughadministration of multiple depot injections near the transplantationsite and/or coating the allograft with artificial particles. In oneembodiment, a skin allograft testing model is chosen due to thesimplicity of the procedure and the ability to use a control syngenicgraft on the same animal. In one embodiment, a heterotopic hearttransplant testing model is chosen, not only due to its relativelysimple procedure and method of data collection, but also because of theconsistency in the rejection profile as opposed to liver and kidneytransplants.

In one embodiment, the present invention contemplates a methodcomprising measuring regulatory T cell migration and activationfollowing exposure to synthetic, biodegradable constructs. In oneembodiment, the method comprises an in vivo murine model. This modelprovides unexpected and unpredictable advantages over traditional invitro models, because, as discussed elsewhere, the behavior of T_(reg)cells in vitro is not believed to be equivalent to that observed invivo. M. Belghith et al., Nat Med 9:1202 (September, 2003); Green etal., PNAS 100:10878 (2003).

In one embodiment, the present invention contemplates a testing methodcomprising co-implanting a tissue transplant graft and a plurality ofartificial particles in vivo, wherein the artificial particles recruitand activate the T_(reg) cells to induce a permanent graft tolerance.For example, artificial particles may be tested for the ability torecruit and stimulate regulatory T-cells by injecting artificialparticles into mice which have been adoptively transferred with GFP+T_(reg) cells. Imaging studies can then easily identify in vivolocalizations of the GFP+ T_(reg) cell following artificial particleinjection. Further, various artificial particle formulations may beco-administered at the time of tissue transplantation (i.e., forexample, skin allograft or heterotopic heart transplantation) to examinetheir therapeutic ability to induce allograft tolerance.

In one embodiment, the present invention contemplates an in vivo methodcomprising manipulating T_(reg) cells using synthetic dendritic cells.In one embodiment, the synthetic dendritic cells stimulate T_(reg),thereby resulting in immunosuppression. Although it is not necessary tounderstand the mechanism of an invention, it is believed that thatsynthetic dendritic cells which are approximately 10 μm do not move fromthe site of implantation given that they are too large to cross theendothelial barrier or redistribute via the reticuloendothelial system.In one embodiment, the method further comprises administering syntheticdendritic cells with encapsulated soluble factors and/or surface factorsinto a first leg flank of a B6 mouse. In one embodiment, theadministering comprises injection. In one embodiment, the injectioncomprises a subcutaneous injection. In one embodiment, the injectioncomprises an intramuscular injection. In one embodiment, a second legflank is injected with a synthetic dendritic cell without encapsulatedsoluble and/or surface factors (i.e., for example, as a negativecontrol). At 1 Day or 3 Days after the injection(s), mice will beeuthanized and skin or muscle will be resected for histological analysis(i.e., for example, staining for T_(reg) cells and Foxp3). Wang et al.,Am J Transplant 6:1297 (June, 2006). The sensitivity of this protocolcan be enhanced by adoptively transferring fluorescent T_(reg) isolatedfrom GFP+B6 mice into normal B6 mice to increase the number ofdetectable cells available for chemotaxis.

In one embodiment, the present invention contemplates a methodcomprising prolonging the survival of a skin allograft using abiomimetic immunosuppressive synthetic dendritic cell.

4. T_(reg) Cells And Transplant Tolerance

The generation of T_(reg) suppressor cells has been attempted in vivoprior to transplantation with the goal of inhibiting allograftrejection. Most of these methods involve the administration of analloantigen, with the final result being an increase in the number andactivation state of regulatory T-cells. In mice, it has been shown thatnaïve regulatory T-cells still have suppressive capacity, but 10 foldgreater quantities are required to promote graft acceptance whencompared to populations that have been stimulated prior totransplantation with alloantigen. Graca et al., J Immunol 168:5558-5565(2002). Furthermore, administration of this alloantigen is usuallyaccompanied by CD4 monoclonal antibodies to avoid eliciting a hostileimmune response. Kingsley et al., J Immunol 168:1080-1086 (2002).

Other monoclonal antibodies which block co-stimulation pathways may alsobe used to generate regulatory T-cells including, but not limited to,monoclonal antibodies that block CD154-CD40 engagement. van Maurik, A.,Herber, M., Wood, K. J. & Jones, N. D. (2002) J Immunol 169, 5401-4;Taylor et al., Blood 99:4601-4609 (2002). Also, CD-3 monoclonalantibodies were used to generate regulatory T-cells via stimulationthrough the T-cell receptor (TCR) and was reported to control theprogression of diabetes. Herold et al., N Engl J Med 346:1692-1698(2002). Native, immature dendritic cells may be capable of generatingCD4+CD25+ T_(reg) cells in vivo if alloantigen is specifically targetedto them by an antibody which binds to DEC205; a receptor involved in therecycling of antigen through late endosomal compartments. Mahnke et al.,Blood 101:4862-4869 (2003).

Ex vivo expansion and manipulation of tolerogenic cells holds muchpromise for therapeutic application given their relatively low frequencyin peripheral tissues. For this potential to be realized, this expansionwould have to maintain an appropriate regulatory phenotype, includingthe ability to migrate to the correct sites of inflammation and thelymphoid compartments. Attempts at generating regulatory T-cells invitro have involved stimulation with alloantigen with co-stimulants,such as: i) vitamin D3 and dexamethasone (Barrat et al., J Exp Med195:603-616 (2002)); ii) IL10 (Levings et al., J Exp Med 193:1295-302(2001)); iii) TGF-β (Yamagiwa et al., J Immunol 166:7282-7289 (2001));and iv) IL2 (Taylor et al. Blood 99:3493-3499 (2002)). Transfection ofT-cell subsets with the gene for FoxP3 in vitro has also endowedregulatory capacity. Fontenot et al., Nat Immunol 4:330-336 (2003).

X. Biomimetic Artificial Antigen Presenting Cells

In one embodiment, the present invention contemplates a methodcomprising fabricating cell-sized, degradable particles (i.e., forexample, a synthetic dendritic cell or artificial antigen presentingcell). In one embodiment, the particles release soluble factors. In oneembodiment, the particles display surface bound factors in a non-randompattern. In one embodiment, the particles display surface bound factorsin a random pattern. In one embodiment, the non-random pattern simulatesan immunological synapse. In one embodiment, the synapse comprises asupramolecular activation complex.

In one embodiment, the artificial particle comprises a biodegradableconstruct thereby providing a controlled release of incorporated and/orattached compounds (i.e., for example, therapeutic agents, antibodies,cytokines, or chemokines). In one embodiment, the particle comprises adegradable polyester including, but not limited to,poly(lactic-co-glycolic) acid (PLGA). PLGA has been used in FDA-approvedgrafts, sutures, and/or drug delivery microparticulates such as LupronDepot®. Degradable PLGA polymer microparticles are superior toconventional latex or polystyrene “artificial APCs” because PLGA confersbiodegradability. Further, unlike latex and polystyrene polymerparticles that only allow surface attachment of proteins, PLGA polymerparticles. allow encapsulation of protein cell factors (i.e., forexample, IL2, TGF-(3, and CCL22) through a double emulsion/solventevaporation procedure. Odonnell et al., Advanced Drug Delivery Reviews28:25-42 (1997). Further, a controlled release of soluble cell factorsfrom PLGA polymers can be engineered to create an appropriate localconcentration of these cell factors, which would be accompanied bycell-to-cell contact with immobilized molecules on the particle surface.Such immobilized molecules (i.e., for example, a monoclonal antibody)can be easily bound to the particle through a streptavidin-biotinlinkage using several established chemical techniques. Further, thecontrolled release of encapsulated one such soluble protein factor fortwenty days from microparticles has been demonstrated (i.e., forexample, CCL22). See, FIG. 9A. Scanning electron microscopy confirmedthe porous nature of the microparticles responsible for the controlledrelease characteristics. See, FIG. 9B.

Artificial stimulation using randomly coated micron-sized sphericalconstructs to promote cytotoxic T-cell receptor engagement along withco-stimulation has been used to gain insight into the role of IL2 as asoluble stimulation factor. Mescher et al., J Immunol 149:2402-2405(1992); and Curtsinger et al., J Immunol Methods 209:47-57 (1997). Whenapplied to regulatory T-cells, stimulation by randomly coated constructsprovided substantial increases in total cell count, but also enhancedsuppressive capabilities over non-stimulated naïve CD4+CD25+ cells.Others have shown that randomly coated particles produce expanded andactivated regulatory T-cells were polyclonal, and maintained expressionof appropriate chemokine receptors. Hoffmann et al., Blood 104:895-903(2004).

Nevertheless, these references using randomly coated particles do notcontemplate the specific advantages of creating and using non-randomlypatterned artificial presenting cells that specifically mimic biologicalcell surface protein patterns (i.e., for example, a supramolecularactivation complex). In one embodiment, the present inventioncontemplates fabricating an artificial tolerogenic dendritic celldesigned to stimulate a regulatory T-cell. In one embodiment, these cellsized constructs present protein factors capable of both cooperating inTCR engagement and releasing T_(reg) cell co-stimulatory moieties. Forexample, the constructs are able to contain and controllably releasesoluble chemokines and proliferation promoting factors. These constructshighly flexible, being capable of modification for both the type, andlevel, of surface bound and/or soluble releasable stimulating factors.In some embodiments, the use of biodegradable materials will provideuses of these “artificial dendritic cells” for in vivo diagnostics toidentify the role of individual T_(reg) cell stimulating factors, andtherapeutically to promote allograft tolerance.

A. The Particle Core

In one embodiment, the present invention contemplates a particlecomprising CD3 antibodies and CD28 antibodies non-randomly attached tothe surface of a poly-lactic-co-glycolic acid (PLGA) particle. In oneembodiment, the antibodies are attached by absorption. In oneembodiment, the antibodies are attached covalently. In one embodiment,the particles are covalently surface modified with streptavidin. In oneembodiment, the particles comprise biotinylated mAb, wherein the mAbsstimulate T_(reg) cells. In one embodiment, the attached antibodies arein a biologically active state. See, FIG. 1.

These biodegradable particles have numerous advantages over T_(reg) cellmodulators known in the art, for example: a) particle biodegradabilityand biocompatibility allow for in vivo investigational and therapeuticuse for controlling T_(reg) cells in immune suppression; or 2) suchparticles are capable of controllably releasing soluble proteins such ascytokines and chemokines from the particle interior while alsopresenting immobilized protein factors on their surface; and 3)surface-mediated stimulation and release of soluble factors from thesame artificial construct closely mimics T_(reg) cell stimulation bybiological dendritic cells.

In one embodiment, the particles comprise releasable soluble factorsthat preferentially activate different T-cell subsets. In oneembodiment, the releasable factors are encapsulated within theparticles. Although it is not necessary to understand the mechanism ofan invention, it is believed that the releasable factors arecontrollably released subsequent to particle degradation. In oneembodiment, the factor comprises a suppressive cytokine (i.e., forexample, TGF-β12). In one embodiment, the factor comprises aT_(reg)-preferential cytokine (i.e., for example, CCL-226). Theseparticles behave similarly to tumor tissues that are believed toactively suppress immune responses by selectively secreting TGF-β12and/or CCL-226 to attract T_(reg)s to their vicinity. Otherwise, abody's normal immune response might otherwise destroy the malignancy.Curiel et al., Nat Med 10:942 (2004); and Chen et al., Proc Natl AcadSci USA 102:419 (2005). Although it is not necessary to understand themechanism of an invention, it is believed that the particlescontemplated by the present invention are capable of both delivering theimmobilize surface stimulation factors and the secreted solublestimulation factors to act in a similar manner as tolerogenic biologicaldendritic cells (i.e., for example, APCs) to influence T_(reg) mediatedimmunoresponsivity. See, FIG. 2.

B. Artificial Antigen Presenting Cell Surface Protein Patterns

Recent studies have attempted to study IS orientations in vitro usingmicrofabricated, patterned “dots” of CD3 antibody (i.e., providing TCRstimulation) in a field of ICAM adhesion molecules. Doh et al., ProcNatl Acad Sci USA 103:5700 (2006). In vitro models have distinctdisadvantages, however, because T_(reg) cell stimulation is morepronounced when using a spherical structure as opposed to a flatsurface, such as a cell culture dish. Curtsinger et al., J ImmunolMethods 209: 47 (1997). Further, the in vitro approach provided inCurtsinger et al. does not allow simulation for the study of all three(3) SMAC regions in a mature IS. Especially lacking in Curtsinger et al.is the ability to study biomimetic SMAC regions. Although recent datademonstrate patterns having protein orientations using two (2) factorson a particle surface, a method of precisely orienting a pattern of >2factors on a particle has yet to be discovered. Chen et al., Proc NatlAcad Sci USA 104:11173 (2007).

In one embodiment, the present invention contemplates a methodcomprising creating a synthetic dendritic cell surface comprising abiomimetic IS pattern. See, FIG. 4. In one embodiment, the IS patterncomprises a cSMAC region. In one embodiment, the IS pattern comprises apSMAC region. In one embodiment, the IS pattern comprises a dSMACregion. Although it is not necessary to understand the mechanism of aninvention, it is believed that this method is analogous to drainingwater out of a tub filled with bowling balls. After only a fraction ofthe liquid has drained, the top portion of the ball (having a circularshape, if viewed from above) will have been exposed (thereby making thesurface available for a first modification) while the remaining portionis still covered by the water (not available for a first modification).After further draining, an annular ring comprising the firstmodification and the remaining portion is exposed, thereby making theremaining portion available for labeling with a second factor, therebycreating a second annular ring. This process may be repeated as manytimes as desired limited only by the size of the ball and the width ofthe multiple annular rings.

A method for patterning a prototype particle with multiple factor layerswas demonstrated using poly-dimethylsiloxane (PDMS). As is known fromphotolithography techniques, PDMS can be etched by the application of asolvent. This is analogous to partial draining of water from a bathtubfull of bowling balls. Using PDMS, a prototype synthetic dendritic cellcomprising a “bull's eye” IS was made by surface etching an artificialcSMAC region surrounded by artificial pSMAC region. See. FIG. 4. In oneembodiment, the present invention contemplates a method for stimulatingT_(reg) cells using “bull's eye” patterned IS-mimetic particles, whereinthe stimulation is qualitatively and quantitatively superior torandomly-labeled synthetic dendritic cells.

The above construction of prototype synthetic dendritic cells using duallabels definitively shows preferential labeling in different particleregions. Nonetheless, some factor overlap is present indicated byoverlapping fluorescent signals (i.e., a yellow central regionindicating the presence of both labels) and some gold-bead labeling inthe SEM images outside of the intended central region. Theseobservations may be a result of uneven PDMS etching which may beimproved by using techniques that are more easily controllable withrespects to speed, distance, and residual matter. For example, a sodiumsilicate (“liquid-glass”) system can provide a more easily controlledetching process by using<5% hydrofluoric acid (HF) evenly applied at acontrollable rate of approximately 100 nm per minute resolution (datanot shown).

C. Functional Protein Patterns on Synthetic Dendritic Cell Prototypes

The present invention contemplates compositions comprising a fabricatedcell-sized, biodegradable particle exhibiting secretable soluble factorsand immobilized surface factors displayed in a biomimetic pattern.

In one embodiment, the present invention contemplates a syntheticdendritic cell comprising PLGA microparticles. In one embodiment, thepresent invention contemplates a method of producing a syntheticdendritic cell comprising encapsulating soluble and/or surface factorsinto the PLGA microparticles using one of many emulsification and/orencapsulation procedures (i.e., for example, double emulsion). In oneembodiment, the synthetic dendritic cells comprise microparticlediameters of approximately 1-1000 μm. In one embodiment, themicroparticle diameters are approximately 5-500 μm. In one embodiment,the synthetic dendritic cells comprise microparticle diameters ofapproximately 10-50 μm. Lu et al., J Biomed Mater Res 50:440 (2000).Encapsulation methods are commonly used to load agents into polymermatrices that are capable of releasing factors by modifying parametersincluding, but not limited to, copolymer ratios and/or molecular weight.Odonnell et al., Advanced Drug Delivery Reviews 28:25 (1997).

Encapsulation loading of synthetic dendritic cells can be easilyverified by dissolution techniques using sodium hydroxide and/ordimethylsulfoxide followed by protein detection assays such asbicinchoninic acid (BCA) or enzyme linked immunosorbent assay (ELISA) todetect total protein. Fluorescent surface factor labeling determineswhich construction method (PDMS vs. “liquid glass”) produces the mostconsistent results. For example, three separate labels can be appliedusing a bioconjugate strategy, for example: i) a first exposed regionmay be protected with small-molecule biotinylation; ii) a second exposedregion may be protected with an asymmetric disulfide (for subsequentthiol exchange) 20; and iii) a third exposed region may be protectedwith standard N-(3-dimethylaminopropyl)-N-ethylcarbodiimidehydrochloride (EDC) chemistry.

In one embodiment, the present invention contemplates a method forevaluating three (3) exposed particle regions by fabricating theparticles using different formulations. In one embodiment, a formulationcomprises cSMAC (CD3 mAb)+pSMAC (ICAM) (2 labels/2 regions) forcomparison to formulations created on a flat surface (i.e., for example,an in vitro cell culture dish). In one embodiment, a formulationcomprises cSMAC (CD3+CD28 mAbs)+pSMAC (ICAM) (3 labels/2 regions) forcomparison with randomly displayed CD3 mAbs and CD28 mAbs particles. Inone embodiment, a formulation comprises cSMAC (CD3/CD28)+pSMAC(ICAM)+dSMAC (CD45 mAb) for comparison with a complete syntheticimmunological synapse comprising at least three (3) SMACs.

XI. Controlled Release of Compounds from Artificial Particles

Controlled release of compounds from microparticles (i.e., for example,PLGA microparticles) fabricated from the double emulsion procedureprovide many advantages as an artificial construct for releasingencapsulated compounds as opposed to more traditional compounds such aslatex or styrene based particles. PLGA microparticle advantages include,but are not limited to: i) the double emulsion procedure allows forencapsulation of any number of water soluble activation factors; ii)particle size can be easily adjusted based on known fabricationparameters; iii) PLGA is non-toxic, biodegradable, and has propertieswhich allow it to be tuned to adjust release of the encapsulated agents;and 4) the surface of PLGA particles can be easily labeled using EDC(carbodiimide) chemistry.

In one embodiment, the present invention contemplates a methodcomprising encapsulating and controllably releasing soluble factorswhile maintaining relatively stable surface presentation of monoclonalantibody. Although it is not necessary to understand the mechanism of aninvention, it is believed that T_(reg) cells are attracted towards anartificial particle, engage surface protein stimulation factors, andreceive the proper proliferation and maturation signals from secretedsoluble proteins at the same time. In one embodiment, the encapsulatingcomprises at least one soluble secretable factors, including but notlimited to, IL2, TGF-β, and CCL22. These factors may be encapsulatedindividually or in any combination. In one embodiment, the releasing iscontrolled by using different molecular weight PLGA or through otherfabrication parameters including, but not limited to, drug distribution,occlusion radius, amorphicity/crytallinity of the polymer, excipientsetc. Rothstein et al., J Materials Chem 18:1873-1880 (2008). Anempirical process determines the final amounts of factors to beencapsulated given that the appropriate quantity of these factors foroptimal stimulation of regulatory T-cells in vivo is yet still unknown.

In one embodiment, the particle surface is covalently labeled todetermine the number of functionalized sites capable of attaching theimmobilized protein factors. In one embodiment, the labeling comprises2-pyridyldithioethylamine. In covalent labeling of the particle, itwould be desirable to allow for positioning of the surface mediatedregulatory T-cell factor(s) (i.e., for example, monoclonal antibodiesfor CD3 or CD28) in a way which does not covalently link the activesite. To achieve this, some embodiments conjugate streptavidin to theparticle surface. Although it is not necessary to understand themechanism of an invention, it is believed that at least one ofstreptavidin's four active binding sites for biotin will be exposed.Consequently, an artificial particle may be labeled using commerciallyavailable biotinylated antibodies specific for many commerciallyavailable surface molecules. Alternatively, streptavidin may bethiolated (i.e., for example, by using readily available products suchas NHS esters of S-acetylthioacetic acid or Trauts reagent) andintegrated into the 2-pyridyldithioethylamine labeling embodimentdescribed herein.

In some embodiments, biodegradable polymers pre-labeled with biotin maybe used in fabrication of artificial particles. For example, poly(lacticacid) (PLA) copolymers with poly(ethylene glycol) (PEG) are reported tobe conjugated with biotin. Cannizzaro et al., Biotechnol Bioeng58:529-535 (1998). Microparticles have been made from these polymershave been reported to produce microparticles having surface bindingsites using emulsion/solvent-evaporation techniques. Further, themicroparticle surface was successfully labeled using an excess ofneutravidin followed by biotinylated monoclonal antibodies and proteins.Sakhalkar et al., Proc Natl Acad Sci USA 100:15895-15900 (2003).

XII. Release Characteristics Of CCL22

The data presented herein demonstrates that a sustained release of CCL22was achieved by loading the chemokine into degradablepoly(lactic-co-glycolic) acid (PLGA)-based microparticles (CCL22MP)using a water-oil-water double emulsion-evaporation technique. Zhao etal., Biomaterials 26:5048-5063 (2005). Scanning electron micrographs(SEM) of intact microparticles indicates that these microparticles arespherical and slightly porous. FIG. 15A. SEM particle cross-sectionsalso show a plurality of internal structures formed during creation in awater-in-oil primary emulsion. FIG. 15B. The surface of a microparticlecomprising CCL22 (CCL22MP) was specifically formulated to be porous, toallow continuous release of chemokine (i.e., for example, releaseoccurring without intermittent periods of lag). Rothstein et al., J.Mater. Chem. 18:1873-1880 (2008); Rothstein et al., Biomaterials30:1657-1664 (2009). For example, release experiments performed inphosphate-buffered saline demonstrate a constant release of CCL22 forover 3 weeks. See, FIG. 16. Further, the microparticles were designed tobe large enough (i.e., for example, >10 μm) to avoid their uptake byphagocytic cells and to prohibit their movement through capillaries,with consequent immobilization at the site of injection. FIG. 17.

Simultaneously, the ability of CCL22 to attract murine T cells wasexamined. Expression of chemokine receptors for CCL22 (i.e., forexample, the CCR4 receptor) are known to control the ability of cells torespond to CCL22 gradients. Moreover, there is evidence that onlyactivated T cells express the CCR4 receptor. Baatar et al., Journal ofImmunology 178: 4891-4900 (2007); Lim et al., Journal of Immunology177:840-851 (2006); and Lee et al., Journal of Experimental Medicine201:1037-1044 (2005); and FIG. 18A. The data presented herein show thatactivated T cells (both regulatory and effector cell populations), butnot naïve T cells, migrated towards a CCL22 gradient in vitro using atranswell chamber-based assay, a response that is consistent with theirsurface receptor expression patterns. FIG. 18B.

Having confirmed that activated Tregs migrate toward a CCL22 gradientand that controlled release formulations could be used to sustain therelease of CCL22, the capability of CCL22MPs to attract Tregs in vivowas tested. For example, CCL22-releasing polymeric particles wereinjected into the triceps surae of FVB mice followed by i.v. infusion ofex vivo activated, alloantigen-specific Tregs (AATregs) thatconstitutively expressed the luciferase gene. Following an activationstimulus (i.e., for example, an allogeneic-dendritic cell injection) themigration pattern of AATregs could be monitored by non-invasive liveanimal imaging. FIG. 19A. The data demonstrate that immediately afterallogeneic-dendritic cell stimulation (i.e., for example, approximately4-7 days after injection) a significantly greater number of AATregs wererecruited to the site of the CCL22MP injection when compared to aninternal control of microparticles lacking CCL22 (BlankMP). FIG. 19B andFIG. 20. Further, upon analyzing the kinetics of migration, it wasdetermined that cellular localization was transient and that within 7days of the stimulation event, no significant numbers of AATregpersisted at the site of particle injections. See, FIG. 19C.

One explanation of the data is that the decline in AATreg numbers may bedue to the absence of stimulatory/inflammatory signals at the site ofCCL22MP injection at that time. Presumably, the presence of Treg can beextended in a stimulatory microenvironment, in combination with othersurvival factors. The potential therapeutic implications of a degradablecontrolled release formulation capable of recruiting Tregs are manifold.For example, CCL22MP may be used in combination with an infusion ofcells expanded ex-vivo. Current pre-clinical data suggest that infusionof freshly-isolated or ex vivo-expanded Tregs can be used to preventrejection of organ transplants and to suppress autoimmune diseases, butchallenges such as obtaining adequate numbers and highly purifiedpopulations of Tregs have hindered progress into clinical trials. Bruskoet al., Immunological Reviews 223:371-390 (2008); and Riley et al.,Immunity 30:656-665 (2009). Given that the present data suggest thatTregs can be attracted to a local site using CCL22, it may be possibleto lower the numbers of injected cells, or potentially, to usepopulations with lower purity. In one embodiment, the present inventioncontemplates a method comprising a microparticle populationencapsulating CCL22, wherein a controlled release of CCL22 from themicroparticle improves tissue transplantation success (i.e., forexample, murine pancreatic islet cell transplantation). In oneembodiment, the released CCL22 attracts Tregs to the pancreatic isletcells Zhang et al., Immunity 30:458-469 (2009).

Another use of CCL22-containing polymeric microparticles would be toattract naturally occurring Treg populations. If the numbers ofrecruited Tregs prove sufficient to control adverse immune responses atthe site of particle injection, then therapeutic effects could berealized without infusion of ex vivo-expanded Tregs. This hypothesis hasbeen reported within the context of a murine model of periodontitis, adisease that is associated with dissipation of Tregs from the gingivaltissues and loss of immune homeostasis. Garlet et al., Microbes andInfection 7:738-747 (2005). The data presented herein indicates thatadministration of CCL22MP leads to local recruitment of FoxP3+ cells tothe gingival tissues and corresponding reversal of adverse outcomesassociated with periodontitis (infra). Conventional methods ofCCL22-based sustained release have disadvantages in that this particularchemokine is known to attract both activated Tregs and activatedeffector T cells. FIG. 18; and Bromley et al., Nature Immunology9:970-980 (2008); Thus, in some cases it is quite possible that thenumbers of recruited Tregs may be insufficient to control the immuneresponse.

However, CCL22-based release formulations can easily be modified tosimultaneously release immunosuppressive agents that can suppress thefunctions of activated effector T cells in situ, thereby assisting theTregs in controlling adverse immune responses. Additionally, althoughCCL22 can attract both activated Tregs and effector T cells in vitro,studies in vivo suggest that CCL22 production and release associatedwith tumors or long-surviving allografts results in localimmunosuppression. Curiel et al., Nature Medicine 10:942-949 (2004); andLee et al., Journal of Experimental Medicine 201:1037-1044 (2005),respectively. Although it is not necessary to understand the mechanismof an invention, it is believed that this CCL22-associated effect may bedue to an enriched population of recruitable Tregs in the periphery whencompared to activated effector T cells, or where equivalent numbers ofboth populations are recruited and the suppressive effect of Tregs isdominant.

In conclusion, the local attraction of Tregs in vivo usingchemokine-loaded sustained delivery vehicles has been demonstrated.Controlled release microparticle formulations as contemplated herein areparticularly useful as a modular platform for therapeutic development aswell as a tool to study Treg-dependent modulation of immune responses insitu.

XIII. CCL22 Microparticle Therapy

To verify that Tregs are recruited toward microparticles that sustainrelease of CCL-22, formulations were administered into the periodontiumof diseased mice. Briefly, mice were orally exposed to the periodontalpathogen A. actinomycetemcomitans (Aar) on day 0, and also receivedCCL-22 microparticles (CCL-22 MPs) or blank (empty) microparticles(BlankMPs) in their periodontal pockets. Specifically, mice receivedmicroparticle injections at day −1, 10 and 20, mice treated with VIPinjections received systemic injections at days −1, 7, 14, 21, 28.Untreated mice served as negative controls. At day 30, all mice weresacrificed and maxilla's were resected. Real-time polymerase chainreaction (PCR) analysis provided strong evidence that our formulationsrecruit Tregs to a greater extent than other groups as detected bygreater expression of FoxP3. FIG. 22A. This same treatment group alsoshowed statistically greater expression of the pro-regenerative cytokineIL-10. Figure BIB. Further, a decreased expression of bone resorbingcell activator RANKL was observed. FIG. 22C. Interestingly, systemic VIPinjections induced endogenous production of CCL-22 presumably leading toTreg recruitment, and the disease symptom attenuation as demonstratedbelow.

To confirm that Treg recruiting formulations attenuate periodontaldisease symptoms, levels of alveolar bone resorption was examined inexperimental mouse periodontitis. G. P. Garlet et al., Clin Exp Immunol147, 128 (January, 2007); D. T. Graves, D. Fine, Y. T. Tang, T. E. VanDyke, G. Hajishengallis, J Clin Periodontol 35, 89 (February, 2008); andJ. J. Yu et al., Blood 109, 3794 (2007). To this end, B6 mice (n=6) wereinfected orally with the periodontal pathogen A. actinomycetemcomitans(Aa) and treated with microparticles containing CCL-22 (CCL-22 MPs) orempty microparticles (BlankMPs) according to the schedule outlinedabove. Additionally vasoactive intestinal peptide (VIP) was administeredintraperitoneally. Mice receiving only PBS and the thickenercarboxymethyl cellulose served as controls (Untreated). Experimentaltreatments were administered in the periodontal pocket of the rightmaxilla while left maxillae were used as internal negative controls ineach mouse. Alveolar bone loss was quantified as the area between thecementoenamel junction (CEJ) and the alveolar bone crest (ABC) FIG. 23,CEJ and FIG. 23A, ABC). Alveolar bone levels are shown on the left(internal control, FIG. 23A LEFT) and right (BlankMP treated, FIG. 23ARIGHT) maxilla. Both the internal control and BlankMP treated maxilla(FIG. 23A, both) exhibited significant alveolar bone loss. However, FIG.23 RIGHT depicts a maxilla treated with CCL-22 MPs, revealingsignificantly reduced alveolar bone loss, both compared to the internalcontrol maxilla (FIG. 28 LEFT) and the BlankMP treated maxilla (FIG. 23ARIGHT). Quantification of total bone loss (area in mm2) reveals thatCCL-22 MP as well as VIP (systemic IP injections) treatments led tosignificantly less alveolar bone resorption than BlankMPs. FIG. 23C.

IVX. Clinical Applications

A. Immunosuppressant Therapy Substitution for Tissue/Graft Transplants

The development of new immunosuppressive agents has been the generaldirection in efforts to improve the transplantation success rate. Forexample, the use of agents which inhibit the production of lymphocyteproliferation factors have extended the half life of pediatric hearttransplants. Pietra et al., Prog Pediatr Cardiol 11:115-129 (2000).Presently, research is underway in efforts to further increase pediatricheart half-lives using agents which inhibit the de novo synthesis ofnucleotides and block co-stimulation of T-cells.

Presently available immunosuppressant drugs act to abrogate acuterejection of tissue transplants. Tissue transplant rejections areusually characterized by recognition of the foreign donor antigen by theadaptive immune system and the subsequent expansion of lymphocytes whichattack the transplanted tissue. Symptoms which accompany acute rejection(including, but not limited to, pain at the transplantation site orfever-like symptoms) are more frequent and severe immediately followingtransplantation, but commonly recur 6-12 months subsequent to theprocedure. Persistent episodes of acute rejection are thought to lead toa state of chronic rejection coupled with a failure of manyimmunosuppressant drugs. The most viable option following the onset ofchronic tissue rejection is re-transplantation, which is undesirablefrom both the standpoint of allograft availability and the increaseddifficulty of the repeated operation.

A major disadvantage of immunosuppressant therapy is that in order toavoid both episodes of acute rejection and the initiation of chronicrejection, immunosuppressant drugs must be administered over the entirelife of the transplant recipient. A further disadvantage ofimmunosuppressant therapy is the frequent incidence of side effects. Forexample, in the case of cyclosporine treatment (a commonly used agentwhich decreases the production of interleukin 2, or IL2), patients havebeen known to experience tremors, headaches, blurry vision, high bloodpressure, and inhibited kidney function. Other commonly usedimmunosuppressants also cause frequent side effects, which range fromgrowth retardation to behavioral instability to neurotoxicity. Besidesthese drug specific side effects, the consequences of long termimmunosuppression in general can be profound including, but not limitedto, increases in the risk of infection, heart disease, diabetes, andcancer.

While a combined administration of the immunosuppressant mycophenolatemofetil along with Vitamin D3 has been reported to generate dendriticcells with a tolerogenic phenotype resulting in increased numbers ofregulatory T-cells in mice, mycophenolate mofetil suffers from the sameadverse side effects as the immunosuppressants mentioned above and hasalso been implicated in causing birth defects in animals. Gregori etal., J Immunol 167:1945-1953 (2001). Furthermore, immunosuppressants cancause inhibition of regulatory T-cells as well as the T-cells thesetherapies intend to impede. Li et al., Nat Med 5:1298-1302 (1999). Dueto the potential of these tolerogenic cells, it is important that agreater understanding of the factors involved in the expansion andemployment of T_(reg) cells in vivo is developed.

The present invention contemplates alternatives to extended immunesystem down-regulation provided by immunosuppressant therapy. Suchalternatives are expected to provide a dramatic improvement over thestate-of-the-art in the clinic. For example, the most desirable of thesealternatives would render the patient's immune system in a state ofcomplete, permanent tolerance to a specific foreign body (i.e., forexample, a single antigen and/or antigen complex). Once this completeand permanent tolerance is attained, no further treatment is necessaryto prevent transplant-related adverse reactions. Furthermore, therecipient's immune system would otherwise function normally, being fullycapable of fighting pathogens and/or stopping the progression of tumorcells. Although it is not necessary to understand the mechanism of aninvention, it is believed that infants up to fourteen (14) months oldinnately have tissue tolerance due to the lack of soluble and/or surfacerejection factors which mediate immediate rejection of the transplant.As the infant's immune system matures, however, this innate tissuetolerance is lost. Fan et al., Nat Med 10, 1227-1233 (2004).

B. Implant Tolerance

Numerous polymeric biomaterials and metal materials are implanted eachyear in human bodies. Among them, drug delivery devices provide localtherapeutic effect for diseases which lack efficient treatments.Controlled release systems are in direct and sustained contact with thetissues, and some of them degrade in situ. Thus, both the materialitself and its degradation products must be devoid of toxicity. Theknowledge and understanding of the criteria and mechanisms determiningthe biocompatibility of biomaterials are therefore of great importance.

The classical tissue response to a foreign material leads to theencapsulation of the implant, which may impair the drug diffusion in thesurrounding tissue and/or cause implant failure. This tissue responsedepends on different factors, especially on the implantation site.Indeed, several organs possess a particular immunological status, whichmay reduce the inflammatory and immune reactions. Among them, thecentral nervous system is of particular interest, since many pathologiesstill need curative treatments.

In one embodiment, the present invention contemplates a method ofinducing implant tolerance by the administration of artificial antigenpresenting cells as described herein.

C. Tissue Healing

In one embodiment, the present invention contemplates using artificialmicroparticles comprising soluble and surface bound factors to inducetissue healing states including, but not limited to bone healing, woundhealing, and disease-induced injury tissue restoration.

1. Bone Healing

Over the past several decades, those having ordinary skill in the arthave been attempting to understand the interactions between the cellsthat mediate the process of bone remodeling and healing. It is believedthat the cells that mediate bone formation (i.e., for example,osteoblasts) may be capable of stimulating osteoclast cells. Osteoclastcells have been reported to resorb and remodel bone. Mundy G. R., Bone24(5 Suppl):35S-38S (1999). Osteoclasts, derived from a hemopoieticmonocyte cell lineage (e.g. in the same cell lineage as dendritic cellsand macrophages), resorb bone using proton and enzymatic secretion.Teitelbaum S. L., Science 289; 1504-1508. It is generally believed thatapproximately 10-15% of human bone is perpetually undergoing remodelingin order for our mineralized tissue to grow and adapt to stress.However, the current evidence suggests that when theosteoblast-osteoclast communication pathway breaks down, osteoscleroticsymptoms may occur including, but not limited to, fractures, severeinfections, blindness, deafness, deformities, or stroke.

Signaling factors (i.e., for example, soluble signaling factors andsurface-bound signaling factors) have been consistently implicated asintermediaries between osteoblasts and osteoclasts. Mundy G. R., Bone24(5 Suppl):35S-38S (1999). See, FIG. 13A. In one embodiment, thepresent invention contemplates a composition comprising anantibody-ligated OSCAR, wherein the antibody/OSCAR composition serves asa co-stimulus with the signaling factors. Merck et al., Journal ofImmunology 176(5):3149-3156 (2006). Although it is not necessary tounderstand the mechanism of an invention, it is believed that theantibody/OSCAR composition may be necessary but not sufficient forosteoclast activation alongside other surface-bound signals (i.e., forexample, RANK-L) and soluble signaling factor MCSF27.

Conversely, it is commonly known that secretion of osteoprotegrin (OPN)(a molecule that inhibits RANK-L signaling) can diminish osteoclasticactivity. Udagawa et al., Endocrinology 141:3478-3484 (2000); and Itohet al., Endocrinology 142:3656-3662 (2001). As dendritic cells mayundergo suppression via apoptosis/anergy (supra), a similar mechanismdownregulating the osteoblast-osteoclast interaction could beapplication to diseases where the degree of osteoclast resorptionexceeds that of osteoblast healing including, but not limited to,osteoporosis or osteopenia.

Finally, has been reported that a balance between bone resorption andhealing may be mediated not only by communication of osteoblasts toosteoclasts, but also by communication of osteoclasts to osteoblasts.Martin et al., Trends in Molecular Medicine 11: 76-81 (2005). Thiscross-talk is commonly referred to as “osteo-coupling”. Although boneresorption processes are believed to release soluble factors embedded inbone matrix that can recruit and differentiate osteoblastic precursors,some studies suggest that osteoclasts still strongly stimulateosteoblasts in the absence of resorption. Although it is not necessaryto understand the mechanism of an invention, it is believed that severalsoluble signaling factors and several surface-bound signaling factorsare likely to be involved.

In one embodiment, the present invention contemplates an artificialbiodegradable osteoblast cell that directly and indirectly interactswith an osteoclast cell. In one embodiment, the artificial osteoblastcell mimics osteo-coupling signaling that results in the modulation ofbone healing. In one embodiment, the artificial osteoblast cell acts asa biomimetic compound by providing surface bound RANK-L signalingfactors, anti-OSCAR antibodies, and a soluble signaling factor (i.e.,for example, MIM-1 and/or MCSF-27). Although it is not necessary tounderstand the mechanism of an invention, it is believed that theartificial osteoblast cell is capable of stimulating osteoclast cells toa greater degree than the “unorganized” forms because of a patternedorganization of the signaling factors. In other embodiments, asurface-bound signaling factor comprises osteoclastic differentiationfactor (ODF). ODF has been reported to act as a co-stimulator withRANK-L. Suda et al., Endocrinology Rev. 13:66-80 (1992). In otherembodiments, OPN is used to suppress osteoclasts. Udagawa et al.,Endocrinology 141:3478-3484 (2000).

In some embodiments, osteo coupling may involve a variety of signalingfactors. In one embodiment, a soluble signaling factor comprises MIM1, achemokine specific to osteoblast precursors. Falany et al., Biochemicaland Biophysical Research Communications 281(1):180-1805 (2001). In oneembodiment, a surface-bound signaling factor comprises EphrinB2, thatappears to stimulate osteogeneic differentiation. Zhao, et al., CellMetabolism 4:111-121 (2006). In one embodiment, a surface-boundsignaling factor comprises TGF-13, believed to stimulate osteoblasts.Pfeilschifter et al., Proceedings of the National Academy of Sciences84:2024-2028 (1987). Although it is not necessary to understand themechanism of an invention, it is believed that cell-sized biodegradableartificial osteoblast cells displaying surface bound signaling factors(i.e., for example, RANK-L, ODF, EphrinB2 and/or TGF-β) on a polymersurface and controllably releasing a soluble signaling factor (i.e., forexample, MIM1 and/or MSCF-27) might act, in combination, as “a syntheticbone surface/synthetic osteoclast” thereby stimulating osteoblastsand/or osteoprecursors to a greater degree than the “unorganized” formsof these factors (i.e., for example, by independent local and/orsystemic administration). See, FIGS. 14A and 14B.

In one embodiment, the present invention contemplates a method of makinga functional artificial osteoblast. In one embodiment, the presentinvention contemplates a method of making a function artificialosteoclast. In one embodiment, the method comprises emulsifying apolymer with a soluble signaling factor to produce a controlled releasemicroparticle. In one embodiment, the controlled release microparticlecomprises surface bound signaling factors. In one embodiment, anartificial osteoblast microparticle comprises a soluble MSCF and surfacebound factors comprising RANK-L, αOSCAR, and/or EPF. See, FIGS. 14A and14B. In one embodiment, an artificial osteoclast microparticle comprisesa soluble MIM1 and surface bound factors EphB4 and/or TGF-β. See, FIGS.15A and 15B. Although it is not necessary to understand the mechanism ofan invention, it is believed that the encapsulation and release of theseagents, and even the ability to precisely pre-program the profile ofrelease may be measured and/or predicted using mathematical modeling ofcontrolled delivery and variation of the microstructure and chemicalstructure of the particles.

In one embodiment, the present invention contemplates a method forexamining combinations of soluble factors (i.e., for example secreted)and surface-bound factors (i.e., for example, immobilized) in vitrousing known differentiation markers and functional assays for osteoblastand osteoclast stimulation. In one embodiment, the method measures thecellular effects via standard co-culture assays. Takahashi et al.,Endocrinology 123: 2600-2601 (1988); and Suda et al., Endocrinology128:1792-1796 (1991). In one embodiment, the method uses assays forosteoblast activity and osteoprecursor differentiation includingstaining for alkaline phosphatase, Von-Kossa, and flow cytometricanalysis. In one embodiment, the method uses assays for calciumphosphate pit-formation and TRAP staining.

In one embodiment, the present invention contemplates a method for invivo osteo-coupling using artificial osteoblast and/or osteoclast cells.In one embodiment, the method utilizes a subcutaneous, mousecraniofacial model. In one embodiment, the model determines the effectsof combinations of multiple soluble and surface-bound factors byintradermal and/or subcutaneous injections of artificial osteoblastand/or osteoclast cells can be made right under the skin, wherein thecells result in contacting a bone surface. Although it is not necessaryto understand the mechanism of an invention, it is believed that theseartificial cells can be made large enough as to not move from the siteof injection. In one embodiment, the effect of these various artificialcell formulations in vivo are determined using software capable ofquantifying bone density and lacunae (resorption) formation inhistological sections of the craniofacial bone tissue (BioQuant,Osteoimage).

2. Tissue Healing

Periodontal disease, or periodontitis, is characterized by destructiveinflammation of the periodontium (i.e., for example, gum tissue,supporting bone, and/or ligaments) and is considered the most pressingoral health concern today. Importantly, this disease affects not onlytooth loss, but also the incidence of cardiovascular disease, kidneydisease, respiratory diseases, diabetes, and even premature childbirth.Seymour, G. J., Ford, P. J., Cullinan, M. P., Leishman, S. & Yamazaki,K. Relationship between periodontal infections and systemic disease.Clin Microbiol Infect 13 Suppl 4, 3-10 (2007); Fisher, M. A. et al.Periodontal disease and other nontraditional risk factors for CKD. Am JKidney Dis 51, 45-52 (2008); Boggess, K. A., Beck, J. D., Murtha, A. P.,Moss, K. & Offenbacher, S. Maternal periodontal disease in earlypregnancy and risk for a small-for-gestational-age infant. Am J ObstetGynecol 194, 1316-1322 (2006); Backes, J. M., Howard, P. A. & Moriarty,P. M. Role of C-reactive protein in cardiovascular disease. AnnPharmacother 38, 110-118 (2004); and Offenbacher, S. & Beck, J. D. Aperspective on the potential cardioprotective benefits of periodontaltherapy. Am Heart J 149, 950-954 (2005). Periodontal disease isstrikingly prevalent in the United States, affecting 34% of individualsover the age of 30, or an estimated 78 million Americans. Garlet, G. P.et al. Regulatory T cells attenuate experimental periodontitisprogression in mice. Journal of Clinical Periodontology 37, 591-600. Itis the number-one cause of tooth loss according to the American DentalAssociation. Beyond the US, periodontal disease is estimated to affectup to 20% of the adult population worldwide. Furthermore, theperiodontal biofilm hosts a wide variety of potentially hazardousbacterial species that can lead to systemic infections and inflammatoryimmune diseases. The most predominant periodontal pathogens,Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis,Tannerella forsythia and Fusobacterium nucleatum, present virulencefactors that have been associated with: 1) systemic infections andcomplications; 2) a 4-fold increase in premature births, 3)anorexia-cachexia syndrome, 4) atherosclerosis, and 5) myocardialinfarction and ischemic stroke. As the correlations betweenperiodontitis and the incidence of these other conditions continue to beelucidated, reducing the prevalence of periodontal disease is asignificant current medical problem and must be solved. The continuingprevalence of periodontal disease is likely perpetuated by amisunderstanding of the disease etiology consonant with currenttherapies that are aimed at removal of these remarkably common bacterialspecies. The current standard of care involves the debridement ofcalculus and plaque, often accompanied by local delivery of anantibiotic such as minocycline (Arestin®). These conventional treatmentstemporarily kill pathogens, but do not protect against inevitable futureinfections nor address the sensitivity that is observed in patients thatare disposed to immune dysfunction. Although invasive bacterial speciesare protagonists of the disease, tissue destruction is mediated by anadverse host inflammatory immune response. Baker, P. J. The role ofimmune responses in bone loss during periodontal disease. Microbes andInfection 2, 1181-1192 (2000); Graves, D. T., Fine, D., Teng, Y. T., VanDyke, T. E. & Hajishengallis, G. The use of rodent models to investigatehost-bacteria interactions related to periodontal diseases. J ClinPeriodontol 35, 89-105 (2008).

In some embodiments, the present invention contemplates a method fortreating inflammation caused by periodontal disease. Although it is notnecessary to understand the mechanism of an invention, it is believedthat as the disease progresses, several populations of lymphocytes arerecruited to the periodontium, guided by local gradients of specificlymphocyte-attracting chemokines. It is further believed that theoverall cytokine milieu produced by these specific populations oflymphocytes that ultimately directs the expression of factors thatpromote hard and soft tissue destruction.

For example, in some embodiments an artificial antigen presenting cellprovides a therapy for inflammation resulting from periodontal gumdisease. Periodontal disease is strikingly prevalent in the UnitedStates (34% of individuals over age 30 or an estimated 78 millionAmericans). Eke et al., “CDC Periodontal Disease Surveillance Project:background, objectives, and progress report” J Periodontol 78:1366-1371(2007). Periodontal disease is a cause of tooth loss according to theAmerican Dental Association and is a current oral health concern. It isbelieved that periodontal disease not only affects tooth loss but alsocontributes to the incidence of cardiovascular disease, diabetes, andrespiratory disease such as pneumonia. Furthermore, women who haveperiodontal disease are over 4 times more likely to give birth to achild prematurely. Boggess et al., “Maternal periodontal disease inearly pregnancy and risk for a small-for-gestational-age infant” Am JObstet Gynecol 194:1316-1322 (2006). Clearly, finding a solution to theproblem of periodontal disease is needed in the art. In: PennsylvaniaDepartment of Health report: “Status of Oral health in Pennsylvania”(2002).

The physiological events associated with periodontal disease are wellcharacterized but the pathologic mechanisms of this disease are unknown.It is believed that periodontal disease affects the composition andintegrity of periodontal structures at the dento-gingival junction,alveolar bone, cementum and periodontal ligament. Further it is believedthat periodontal disease causes the destruction of connective tissuematrix and cells, loss of fibrous attachment, and resorption of alveolarbone. FIG. 11. Although it is not necessary to understand the mechanismof an invention, it is believed that tissue destruction in periodontalpatients is thought to be a result of a complex inflammatory and immuneresponse initiated and perpetuated by gram-negative anaerobic rods andspirochetes. Previous work has suggested that B cells and T cellsaccumulate in large numbers in the periodontal tissues although muchabout their functions in the disease process is not clearly understood.

Current therapies for periodontal disease focus upon reducing orcontrolling bacterial infection in the gums. Traditional, mechanicaldebridement techniques temporarily remove the accumulated bacterialplaque responsible for inflammation. One treatment strategy provides acontrolled release of the antibiotic minicycline from PLGA microspheresthat are placed in the periodontal pocket (Arestin®, OraPharma).Although both of these techniques may temporarily reduce inflammationassociated with bacteria, they do not intend to treat the susceptibilityof the area to inflammation and the reformation of pockets thatcultivate further infection leading to re-establishment of disease.Since periodontal tissue destruction is initiated and exacerbated byinflammatory host response, there has been an increased focus towardunderstanding the cellular immune responses in the periodontal space inorder to treat the source of the physiological response.

Recently, several groups have discovered evidence that the cause ofperiodontal disease may related to the regulation of inflammation in thelocal tissues. Cardoso et al., “Characterization of CD4+CD25+ naturalregulatory T cells in the inflammatory infiltrate of human chronicperiodontitis” J Leukoc Biol (2008); and Ernst et al., “Diminishedforkhead box P3/CD25 double-positive T regulatory cells are associatedwith the increased nuclear factor-kappaB ligand (RANK-L+) T cells inbone resorption lesion of periodontal disease” Clin Exp Immunol148:271-280 (2007). It was reported that cytokines which may inhibitand/or regulate inflammation (i.e., for example, IL10) are substantiallydiminished in tissues with periodontal disease. Although it is notnecessary to understand the mechanism of an invention, it is believedthat the absence of these anti-inflammatory cytokines not only result inincreased inflammation, but also produces a cascade leading to theproduction of RANK-L. Although it is not necessary to understand themechanism of an invention, it is believed that RANK-L comprises a factorthat differentiates monocyte precursors (i.e., for example, osteoblasts)into bone-resorbing cells called osteoclasts.

The regulation of anti-inflammatory cytokines and more generally, theregulation of the resultant harmful autoimmune responses, may bemediated by immunosuppressive T-cells (i.e., for example, T_(reg)cells). Tang et al., “The Foxp3+ regulatory T cell: a jack of alltrades, master of regulation” Nat Immunol 9:239-244 (2008). For example,it has been reported that while markers for T_(reg) cells were presentin healthy tissues, they were absent in diseased periodontal tissues.Further, it has been reported that patients with rheumatoid arthritis(predisposition for autoimmune regulation breakdown) have 8-foldincreased odds of developing periodontal disease. Pischon et al.,“Association among rheumatoid arthritis, oral hygiene, andperiodontitis” J Periodontol 79:979-986 (2008).

As discussed above, periodontal disease is currently treated withstrategies focused only on the removal of invasive bacterial species.Specifically, the clinical procedure called scaling and root planinginvolves the mechanical removal of plaque and bacteria from beneath thegingiva (i.e., for example, debridement), and is typically performed bya periodontal specialist. In severe cases, antibiotic treatments in theform of controlled release microparticles may be injected into theperiodontal pocket (i.e., for example, Arrestin®, PLGA microparticlesencapsulating and controllably releasing the antibiotic minocycline for21 days). Williams, R. C. et al. Treatment of periodontitis by localadministration of minocycline microspheres: A controlled trial. Journalof Periodontology 72, 1535-1544 (2001). This treatment approach isinsufficient because, antibiotic treatments only temporarily removes thebacterial species and recurrent infections are common, requiringpatients to repetitively undergo these expensive procedures. While suchantibiotic treatments temporarily remove the bacterial insult, they donot address the sensitivity seen in patients that are susceptible tosuch immune-mediated tissue destruction. Furthermore, antibiotictreatment and/or scaling is completely ineffective in approximately 20%of the population and may induce antibiotic resistance, thereby limitingpatient options for future treatment.

Recent periodontal disease research has investigated the inflammatoryimmune reaction itself. For example, one recent experimental treatmentaimed at reducing the host inflammatory response involves theadministration of the drug Resolvin®. Resolvin® blocksneutrophil-mediated inflammation and the associated pro-inflammatorycytokine milieu. Hasturk, H. et al. Resolvin E1 regulates inflammationat the cellular and tissue level and restores tissue homeostasis invivo. J Immunol 179, 7021-7029 (2007). However, it has been shown thatdirect, long-term, inhibition of inflammatory cytokines by traditionalblocking strategies (i.e., for example, anti-inflammatory compounds) cancompromise periodontal tissue healing. Gurgel, B. C. et al. SelectiveCOX-2 inhibitor reduces bone healing in bone defects. Brazilian oralresearch 19, 312-316 (2005); Ribeiro, F. V. et al. Selectivecyclooxygenase-2 inhibitor may impair bone healing around titaniumimplants in rats. Journal of Periodontology 77, 1731-1735 (2006); Simon,A. M., Manigrasso, M. B. & O'Connor, J. P. Cyclo-oxygenase 2 function isessential for bone fracture healing. Journal of Bone and MineralResearch 17, 963-976 (2002); Vuolteenaho, K., Moilanen, T. & Moilanen,E. Non-steroidal anti-inflammatory drugs, cyclooxygenase-2 and the bonehealing process. Basic and Clinical Pharmacology and Toxicology 102,10-14 (2008); Zhang, X., Kohli, M., Zhou, Q., Graves, D. T. & Amar, S.Short- and long-term effects of IL-1 and TNF antagonists on periodontalwound healing. Journal of Immunology 173, 3514-3523 (2004). Further, ithas also been shown that traditional blocking of the protective immuneresponses actually results in increased bacterial burden and acutesystemic reactions. Garlet, G. P. et al. The essential role of IFN-gammain the control of lethal Aggregatibacter actinomycetemcomitans infectionin mice. Microbes Infect 10, 489-496 (2008); and Garlet, G. P. et al.The dual role of p55 tumour necrosis factor-α receptor in Actinobacillusactinomycetemcomitans-induced experimental periodontitis: Hostprotection and tissue destruction. Clinical and Experimental Immunology147, 128-138 (2007). Consequently, an untested strategy toward diseaseresolution may not require complete blocking of the protective immuneresponses and would clearly be a more desirable approach.

Notably, harmful inflammatory responses are not regulated by blockingleukocyte infiltration, but rather by balancing inflammatory leukocyterecruitment with regulatory lymphocyte recruitment. Vignali, D. A. A.,Collison, L. W. & Workman, C. J. How regulatory T cells work. NatureReviews Immunology 8, 523-532 (2008); and Campanelli, A. P. et al.CD4+CD25+ T cells in skin lesions of patients with cutaneousleishmaniasis exhibit phenotypic and functional characteristics ofnatural regulatory T cells. J Infect Dis 193, 1313-1322 (2006).Regulatory T-cells (Treg) may exert their control over other lymphocytesboth by several mechanisms including but not limited to secretingfactors and through direct cell-cell interactions, ultimately leading totargeted inflammatory-immune cell arrest. However, progress has beenmade toward utilizing Treg therapeutically for a wide variety ofautoimmune and inflammatory diseases. Adoptive (Treg) cell transfer(i.e. cell infusion therapies) have seen the most pre-clinical success.Yet clinical translation of the complicated ex vivo cellular expansionprotocols has proven difficult. Riley, J. L., June, C. H. & Blazar, B.R. Human T Regulatory Cell Therapy: Take a Billion or So and Call Me inthe Morning. Immunity 30, 656-665 (2009). One may speculate thatperiodontal disease treatment would have the best clinical impact withan off-the-shelf therapeutic that improves the body's natural mechanismsfor immune regulation (recruitment/activation of endogenous Treg) inattempt to restore local immune homeostasis and balance.

Naturally, immune cells are recruited to peripheral sites via chemokinessecreted by tissue resident cells. Specifically, biological gradients ofchemokines direct immune cells toward the origin of secretion (i.e., forexample, at an infection site). For example, one way in which tumorsappear to avoid immune surveillance and clearance is through therecruitment of regulatory T cells. Specifically, tumors produce andsustain a biological gradient of CCL22 (a Treg-associated chemokine)that directs Treg migration. Dutzan, N., Gamonal, J., Silva, A., Sanz,M. & Vernal, R. Over-expression of forkhead box P3 and its associationwith receptor activator of nuclear factor-s B ligand, interleukin(IL)-17, IL-10 and transforming growth factor-β during the progressionof chronic periodontitis. Journal of Clinical Periodontology 36, 396-403(2009); Sather, B. D. et al. Altering the distribution of Foxp3+regulatory T cells results in tissue-specific inflammatory disease.Journal of Experimental Medicine 204, 1335-1347 (2007); Garlet, G. P.,Avila-Campos, M. J., Milanezi, C. M., Ferreira, B. R. & Silva, J. S.Actinobacillus actinomycetemcomitans-induced periodontal disease inmice: patterns of cytokine, chemokine, and chemokine receptor expressionand leukocyte migration. Microbes Infect 7, 738-747 (2005). Onceco-localized, Treg suppress effector immune cells by secreting factorssuch as IL-10 and TGF-β, thereby establishing immunological homeostasisin an milieu that would otherwise present itself as highly inflammatory.Rabinovich, G. A., Gabrilovich, D. & Sotomayor, E. M. in Annual Reviewof Immunology, Vol. 25 267-296 (2007).

Although it is not necessary to understand the mechanism of aninvention, it is believed that engineering principles may result in theproduction of controlled release systems that can produce a stablebiological gradient of certain chemokines (i.e., for example, CCL22).These microparticles can be fabricated using an FDA-approved polyesterand will degrade in a well characterized manner in vivo. Rothstein, S.N., Federspiel, W. J., Little, S. R. A unified mathematical model forthe prediction of controlled release from surface and bulk erodingpolymer matrices. Biomaterials (2009); Garlet, G. P. et al. The dualrole of p55 tumour necrosis factor-alpha receptor in Actinobacillusactinomycetemcomitans-induced experimental periodontitis: hostprotection and tissue destruction. Clin Exp Immunol 147, 128-138 (2007).Theoretically, by mimicking the natural immune-evasion mechanisms oftumors using rationally-designed, CCL22-releasing, polymericmicroparticles, Tregs may be recruited to a site of destructiveinflammation (i.e., for example, a diseased periodontium).

In one embodiment, the present invention contemplates a methodcomprising a controlled delivery of Treg chemoattractants (i.e., forexample, CCL22 and/or vasoactive intestinal peptide, VIP) in theperiodontium that can abrogate periodontal disease symptoms. In oneembodiment, the method is clinically viable, biocompatible, andnon-inflammatory. Rothstein, S. N., Federspiel, W. J., and Little, S. R.A simple model framework for the prediction of controlled release frombulk eroding polymer matrices. Journal of Materials Chemistry 18,1873-1880 (2008); Rothstein, S. N., Federspiel, W. J., Little, S. R. Aunified mathematical model for the prediction of controlled release fromsurface and bulk eroding polymer matrices. Biomaterials (2009); Delgado,M., Gonzalez-Rey, E. & Ganea, D. VIP/PACAP preferentially attract Th2effectors through differential regulation of chemokine production bydendritic cells. FASEB Journal 18, 1453-1455 (2004); Little, S. R. etal. Poly-beta amino ester-containing microparticles enhance the activityof nonviral genetic vaccines. Proc Natl Acad Sci USA 101, 9534-9539(2004). In one embodiment, the method comprises a controlled releaseformulation that is tunable and provides a long-lasting delivery of adrug. Although it is not necessary to understand the mechanism of aninvention, it is believed that these embodiments provide a potentialsolution for immune imbalance and dysfunction associated withperiodontal disease, while avoiding immune-blocking strategies. It isfurther believed that these embodiments are supported by preliminarydata showing: 1) Tregs are attracted toward a preliminary formulation ofCCL22 microparticles, in vitro and in vivo; 2) administration of CCL22microparticles results in resolution of periodontal disease symptoms inin vivo animal models; 3) transformation/proliferation of CD4+lymphocyte populations into FoxP3+ regulatory lymphocytes is possibleusing factors including, but not limited to, TGF-β, IL2, and/orrapamycin.

Inflammatory imbalances may cause periodontal tissue destruction whereinthe inflammatory cascade is primarily initiated by bacterial recognitionthrough innate immune cells bearing toll like receptors (TLRs). Garlet,G. P., Avila-Campos, M. J., Milanezi, C. M., Ferreira, B. R. & Silva, J.S. Actinobacillus actinomycetemcomitans-induced periodontal disease inmice: Patterns of cytokine, chemokine, and chemokine receptor expressionand leukocyte migration. Microbes and Infection 7, 738-747 (2005);McGinity, J. W. & O'Donnell, P. B. Preparation of microspheres by thesolvent evaporation technique. Adv Drug Deliv Rev 28, 25-42 (1997);Burns, E., Bachrach, G., Shapira, L. & Nussbaum, G. Cutting edge: TLR2is required for the innate response to Porphyromonas gingivalis:Activation leads to bacterial persistence and TLR2 deficiency attenuatesinduced alveolar bone resorption. Journal of Immunology 177, 8296-8300(2006); Kajita, K. et al. Quantitative messenger RNA expression ofToll-like receptors and interferon-alpha1 in gingivitis andperiodontitis. Oral Microbiol Immunol 22, 398-402 (2007); Nakamura, H.et al. Lack of Toll-like receptor 4 decreases lipopolysaccharide-inducedbone resorption in C3H/HeJ mice in vivo. Oral Microbiology andImmunology 23, 190-195 (2008). Triggering of these pathogen receptorsmay lead to the induction of pro-inflammatory mediators including, butnot limited to, TNF-α, IL-1β and IFN-Y. Garlet, G. P., Martins Jr, W.,Ferreira, B. R., Milanezi, C. M. & Silva, J. S. Patterns of chemokinesand chemokine receptors expression in different forms of humanperiodontal disease. Journal of Periodontal Research 38, 210-217 (2003);Garlet, G. P. et al. Cytokine pattern determines the progression ofexperimental periodontal disease induced by Actinobacillusactinomycetemcomitans through the modulation of MMPs, RANKL, and theirphysiological inhibitors. Oral Microbiology and Immunology 21, 12-20(2006). This skewed, pro-inflammatory environment may upregulate softtissue destroying matrix metalloproteinases (MMPs) and/or RANKL areceptor activator of nuclear factor κB ligand and a potent activator ofbone resorbing osteoclasts. Garlet, G. P. et al. The essential role ofIFN-gamma in the control of lethal Aggregatibacter actinomycetemcomitansinfection in mice. Microbes Infect 10, 489-496 (2008); Garlet, G. P. etal. The dual role of p55 tumour necrosis factor-α receptor inActinobacillus actinomycetemcomitans-induced experimental periodontitis:Host protection and tissue destruction. Clinical and ExperimentalImmunology 147, 128-138 (2007); Tang, Q. & Bluestone, J. A. The Foxp3+regulatory T cell: A jack of all trades, master of regulation. NatureImmunology 9, 239-244 (2008); Normenmacher, C. et al. DNA fromperiodontopathogenic bacteria is immunostimulatory for mouse and humanimmune cells. Infection and Immunity 71, 850-856 (2003); Graves, D. T. &Cochran, D. The contribution of interleukin-1 and tumor necrosis factorto periodontal tissue destruction. J Periodontol 74, 391-401 (2003).Inflammatory mediators may result in tissue destruction in theperiodontium and inhibit the production of anti-inflammatory factorsconducive for disease amelioration and healing. Consequently, thepresent invention contemplates a method for solving the periodontaldisease problem by controlling the inflammatory response associated withthe known immunological imbalance associated with periodontitis.

Tregs have been shown to reestablish immune homeostasis through a widevariety of mechanisms, both at the site of inflammation (i.e., forexample, the periodontium) and/or at the draining lymph nodes (i.e., forexample, the cervical lymph nodes). Taubman, M. A., Valverde, P., Han,X. & Kawai, T. Immune response: the key to bone resorption inperiodontal disease. J Periodontol 76, 2033-2041 (2005) Specifically,Tregs act to balance these pro-inflammatory mediators by secretinganti-inflammatory factors including, but not limited to, IL-10 and/orTGF-β. Vignali, D. A. A., Collison, L. W. & Workman, C. J. Howregulatory T cells work. Nature Reviews Immunology 8, 523-532 (2008);Cardoso, C. R. et al. Characterization of CD4+CD25+ natural regulatory Tcells in the inflammatory infiltrate of human chronic periodontitis.Journal of Leukocyte Biology 84, 311-318 (2008). Indeed, recent reportshave shown that IL-10 levels are substantially diminished in patientswith severe periodontitis. Zhang, N. et al. Regulatory T CellsSequentially Migrate from Inflamed Tissues to Draining Lymph Nodes toSuppress the Alloimmune Response. Immunity 30, 458-469 (2009). IL-10 hasbeen shown to play a major role in attenuation of periodontal disease byupregulating an extracellular RANKL inhibitor osteoprotegerin (OPG) andpromoting increased levels of intra-inflammatory-cell ‘suppressors ofcytokine signaling’ (SOCS). Claudino, M. et al. The broad effects of thefunctional IL-10 promoter-592 polymorphism: Modulation of IL-10, TIMP-3,and OPG expression and their association with periodontal diseaseoutcome. Journal of Leukocyte Biology 84, 1565-1573 (2008); Garlet, G.P., Cardoso, C. R., Campanelli, A. P., Martins Jr, W. & Silva, J. S.Expression of suppressors of cytokine signaling in diseased periodontaltissues: A stop signal for disease progression? Journal of PeriodontalResearch 41, 580-584 (2006). Furthermore, IL-10 not only regulates theinflammatory immune response, but also plays a key role in boneanabolism, leading to maturation of bone forming osteoblasts. Abe, T. etal. Osteoblast differentiation is impaired in SOCS-1-deficient mice. JBone Miner Metab 24, 283-290 (2006); Lorentzon, M., Greenhalgh, C. J.,Mohan, S., Alexander, W. S. & Ohlsson, C. Reduced bone mineral densityin SOCS-2-deficient mice. Pediatr Res 57, 223-226 (2005); Ouyang, X. etal. SOCS-2 interferes with myotube formation and potentiates osteoblastdifferentiation through upregulation of JunB in C2C12 cells. J CellPhysiol 207, 428-436 (2006). Finally, TGF-β has been shown to play animportant role in immune regulation and tissue healing, specificallythrough the recruitment and guidance of periodontal ligament cells.Ernst, C. W. O. et al. Diminished forkhead box P3/CD25 double-positive Tregulatory cells are associated with the increased nuclear factor-kBligand (RANKL+) T cells in bone resorption lesion of periodontaldisease. Clinical and Experimental Immunology 148, 271-280 (2007).

In one embodiment, the present invention contemplates a method forrestoring the immunosupressive regulatory balance in periodontal space,thereby reversing periodontal disease progression. In one embodiment,the population of Treg cells in the periodontal space is increased. Inone embodiment, the amount of immunosupressive cytokines (i.e., forexample, CCL-22) in the periodontal space is increased. Although it isnot necessary to understand the mechanism of an invention, it isbelieved that immunosupressive cytokines activate T_(reg) cells thatactually mediate the immunosuppression.

Treg populations have been identified in periodontal lesions. In oneembodiment, the present invention contemplates a method providing acontrolled release of soluble T_(reg) stimulatory factors (i.e., forexample, CCL-22) from soluble biodegradable microparticles afterinjection into a periodontal space. The data presented hereindemonstrates a sustained release of CCL-22 over a period of 1 month frommicroparticles composed of the FDA-approved polymer, PLGA. FIG. 9A.Although it is not necessary to understand the mechanism of aninvention, it is believed that the released cytokine recruits T_(reg)cells back into the periodontal space, thereby restoring immuneregulation and abrogating periodontal disease progression. See, ExampleXII.

In one embodiment, the present invention contemplates a method for thecontrolled delivery of Treg chemoattractants in the periodontium therebypromoting localization of endogenous Treg and abrogating periodontaldisease symptoms. In one embodiment, the method further comprisesrecruiting endogenous Treg to the periodontium. In one embodiment, theperiodontal disease symptoms are abrogated by reducing inflammation.Although it is not necessary to understand the mechanism of aninvention, it is believed that the this method is supported bypreliminary data demonstrating that: 1) controlled release of CCL22leads to the recruitment of endogenous, Foxp3+ regulatory T-cells to theperiodontium; 2) administration of CCL22 controlled release formulationsleads to both higher numbers of Treg in draining lymph nodes andresolution of periodontal disease symptoms in in vivo animal models; and3) beyond recruitment of Treg, a combination of specific factors havebeen identified that could induce the proliferation and/ortransformation of local CD4+ lymphocytes toward enriched Tregpopulations.

In one embodiment, the present invention contemplates formulationscapable of a long-lasting release of Treg-inducing factors. In oneembodiment, the formulation is used for the treatment of periodontitis.In one embodiment, microparticles comprising CCL22 and/or vasoactiveintestinal peptide (VIP), are constructed of biodegradablepoly(lactic-co-glycolic acid) (PLGA) polymer. Rothstein, S. N.,Federspiel, W. J., and Little, S. R. A simple model framework for theprediction of controlled release from bulk eroding polymer matrices.Journal of Materials Chemistry 18, 1873-1880 (2008). In one embodiment,the PLGA microparticles facilitate Treg recruitment to the periodontium.In one embodiment, the microparticle is a controlled releasemicroparticle.

In one embodiment, the present invention contemplates a methodcomprising a microparticle formulation for treating periodontal disease,wherein the formulation promotes Treg recruitment, thereby reducingalveolar bone loss. Although it is not necessary to understand themechanism of an invention, it is believed that local lymphocytes mayexpand toward an enriched population of Tregs through controlled releaseof a combination of several key molecules. Although it is not necessaryto understand the mechanism of an invention, it is believed thatrecruited Tregs may play a role in periodontal disease abrogation andhost response. For example, CCL22 microparticle formulation and systemicVIP effectively induce Treg migration to periodontal tissues and mayabrogate disease symptoms. In one embodiment, the present inventioncontemplates formulations that induce Treg chemotaxis and therapeuticfunction in the periodontium by monitoring Treg residence and diseasesymptoms. For example, normal mice may be compared to mice deficient inreceptors thought to play a role in chemotactic migration andimmunological function. Further, gene expression levels of Treg markers,inflammatory mediators, soft and hard tissue destroying factors andbiomolecules involved in bone growth to elucidate the mechanisms ofTreg-mediated periodontal disease abrogation may also be assessed.

XV. Antibodies

The present invention provides for the use of antibodies (i.e., forexample, polyclonal or monoclonal). In one embodiment, the presentinvention provides monoclonal antibodies that specifically bind to apolypeptide residing on a T_(reg) cell.

An antibody against a protein of the present invention may be anymonoclonal or polyclonal antibody, as long as it can recognize theprotein. Antibodies can be produced by using a protein of the presentinvention as the antigen according to a conventional antibody orantiserum preparation process.

The present invention contemplates the use of both monoclonal andpolyclonal antibodies. Any suitable method may be used to generate theantibodies used in the methods and compositions of the presentinvention, including but not limited to, those disclosed herein. Forexample, for preparation of a monoclonal antibody, protein, as such, ortogether with a suitable carrier or diluent is administered to an animal(e.g., a mammal) under conditions that permit the production ofantibodies. For enhancing the antibody production capability, completeor incomplete Freund's adjuvant may be administered. Normally, theprotein is administered once every 2 weeks to 6 weeks, in total, about 2times to about 10 times. Animals suitable for use in such methodsinclude, but are not limited to, primates, rabbits, dogs, guinea pigs,mice, rats, sheep, goats, etc.

For preparing monoclonal antibody-producing cells, an individual animalwhose antibody titer has been confirmed (e.g., a mouse) is selected, and2 days to 5 days after the final immunization, its spleen or lymph nodeis harvested and antibody-producing cells contained therein are fusedwith myeloma cells to prepare the desired monoclonal antibody producerhybridoma. Measurement of the antibody titer in antiserum can be carriedout, for example, by reacting the labeled protein, as describedhereinafter and antiserum and then measuring the activity of thelabeling agent bound to the antibody. The cell fusion can be carried outaccording to known methods, for example, the method described by Koehlerand Milstein (Nature 256:495 [1975]). As a fusion promoter, for example,polyethylene glycol (PEG) or Sendai virus (HVJ), preferably PEG is used.

Examples of myeloma cells include NS-1, P3Ul, SP2/0, AP-1 and the like.The proportion of the number of antibody producer cells (spleen cells)and the number of myeloma cells to be used is preferably about 1:1 toabout 20:1. PEG (preferably PEG 1000-PEG 6000) is preferably added inconcentration of about 10% to about 80%. Cell fusion can be carried outefficiently by incubating a mixture of both cells at about 20° C. toabout 40° C., preferably about 30° C. to about 37° C. for about 1 minuteto 10 minutes.

Various methods may be used for screening for a hybridoma producing theantibody (e.g., against a tumor antigen or autoantibody of the presentinvention). For example, where a supernatant of the hybridoma is addedto a solid phase (e.g., microplate) to which antibody is adsorbeddirectly or together with a carrier and then an anti-immunoglobulinantibody (if mouse cells are used in cell fusion, anti-mouseimmunoglobulin antibody is used) or Protein A labeled with a radioactivesubstance or an enzyme is added to detect the monoclonal antibodyagainst the protein bound to the solid phase. Alternately, a supernatantof the hybridoma is added to a solid phase to which ananti-immunoglobulin antibody or Protein A is adsorbed and then theprotein labeled with a radioactive substance or an enzyme is added todetect the monoclonal antibody against the protein bound to the solidphase.

Selection of the monoclonal antibody can be carried out according to anyknown method or its modification. Normally, a medium for animal cells towhich HAT (hypoxanthine, aminopterin, thymidine) are added is employed.Any selection and growth medium can be employed as long as the hybridomacan grow. For example, RPMI 1640 medium containing 1% to 20%, preferably10% to 20% fetal bovine serum, GIT medium containing 1% to 10% fetalbovine serum, a serum free medium for cultivation of a hybridoma(SFM-101, Nissui Seiyaku) and the like can be used. Normally, thecultivation is carried out at 20° C. to 40° C., preferably 37° C. forabout 5 days to 3 weeks, preferably 1 week to 2 weeks under about 5% CO2gas. The antibody titer of the supernatant of a hybridoma culture can bemeasured according to the same manner as described above with respect tothe antibody titer of the anti-protein in the antiserum.

Separation and purification of a monoclonal antibody (e.g., against acancer marker of the present invention) can be carried out according tothe same manner as those of conventional polyclonal antibodies such asseparation and purification of immunoglobulins, for example,salting-out, alcoholic precipitation, isoelectric point precipitation,electrophoresis, adsorption and desorption with ion exchangers (e.g.,DEAE), ultracentrifugation, gel filtration, or a specific purificationmethod wherein only an antibody is collected with an active adsorbentsuch as an antigen-binding solid phase, Protein A or Protein G anddissociating the binding to obtain the antibody.

Polyclonal antibodies may be prepared by any known method ormodifications of these methods including obtaining antibodies frompatients. For example, a complex of an immunogen (an antigen against theprotein) and a carrier protein is prepared and an animal is immunized bythe complex according to the same manner as that described with respectto the above monoclonal antibody preparation. A material containing theantibody against is recovered from the immunized animal and the antibodyis separated and purified.

As to the complex of the immunogen and the carrier protein to be usedfor immunization of an animal, any carrier protein and any mixingproportion of the carrier and a hapten can be employed as long as anantibody against the hapten, which is crosslinked on the carrier andused for immunization, is produced efficiently. For example, bovineserum albumin, bovine cycloglobulin, keyhole limpet hemocyanin, etc. maybe coupled to an hapten in a weight ratio of about 0.1 part to about 20parts, preferably, about 1 part to about 5 parts per 1 part of thehapten.

In addition, various condensing agents can be used for coupling of ahapten and a carrier. For example, glutaraldehyde, carbodiimide,maleimide activated ester, activated ester reagents containing thiolgroup or dithiopyridyl group, and the like find use with the presentinvention. The condensation product as such or together with a suitablecarrier or diluent is administered to a site of an animal that permitsthe antibody production. For enhancing the antibody productioncapability, complete or incomplete Freund's adjuvant may beadministered. Normally, the protein is administered once every 2 weeksto 6 weeks, in total, about 3 times to about 10 times.

The polyclonal antibody is recovered from blood, ascites and the like,of an animal immunized by the above method. The antibody titer in theantiserum can be measured according to the same manner as that describedabove with respect to the supernatant of the hybridoma culture.Separation and purification of the antibody can be carried out accordingto the same separation and purification method of immunoglobulin as thatdescribed with respect to the above monoclonal antibody.

The protein used herein as the immunogen is not limited to anyparticular type of immunogen. For example, a protein expressed resultingfrom a virus infection (further including a gene having a nucleotidesequence partly altered) can be used as the immunogen. Further,fragments of the protein may be used. Fragments may be obtained by anymethods including, but not limited to expressing a fragment of the gene,enzymatic processing of the protein, chemical synthesis, and the like.

XVI. Artificial Antigen Presenting Cell Kits

In another embodiment, the present invention contemplates kits for thepractice of the methods of this invention. The kits preferably includeone or more containers containing an artificial antigen presenting cellof this invention. The kit can optionally include a soluble T_(reg)stimulation protein. The kit can optionally include a surface T_(reg)stimulation protein. The kit can optionally include nucleic acids suchas FoxP3. The kit can optionally include chemicals capable offunctionalizing the polymer ends of an artificial antigen presentingcell. The kit can optionally include a pharmaceutically acceptableexcipient and/or a delivery vehicle (e.g., a liposome). The reagents maybe provided suspended in the excipient and/or delivery vehicle or may beprovided as a separate component which can be later combined with theexcipient and/or delivery vehicle. The kit may optionally containadditional therapeutics to be co-administered with an artificial antigenpresenting cell.

The kits may also optionally include appropriate systems (e.g. opaquecontainers) or stabilizers (e.g. antioxidants) to prevent degradation ofthe reagents by light or other adverse conditions.

The kits may optionally include instructional materials containingdirections (i.e., protocols) providing for the use of the reagents inthe induction and maintenance of tissue tolerance. While theinstructional materials typically comprise written or printed materialsthey are not limited to such. Any medium capable of storing suchinstructions and communicating them to an end user is contemplated bythis invention. Such media include, but are not limited to electronicstorage media (e.g., magnetic discs, tapes, cartridges, chips), opticalmedia (e.g., CD ROM), and the like. Such media may include addresses tointerne sites that provide such instructional materials.

XVII. Pharmaceutical Compositions

The present invention further provides pharmaceutical compositions(e.g., comprising the artificial antigen presenting cells describedabove). The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary (e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances that increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product.

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions.Thus, for example, the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Dosing is dependent on type and amount of tissue that is transplanted.The course of treatment lasting from several days to several months, orin some cases, for the lifetime of the recipient. Optimal dosingschedules can be calculated from measurements of compound accumulationin the body of the patient. The administering physician can easilydetermine optimum dosages, dosing methodologies and repetition rates.Optimum dosages may vary depending on the relative potency of individualoligonucleotides, and can generally be estimated based on EC₅₀, found tobe effective in in vitro and in vivo animal models or based on theexamples described herein. In general, dosage is from 0.01 μg to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly. The treating physician can estimate repetition ratesfor dosing based on measured residence times and concentrations of thedrug in bodily fluids or tissues.

EXPERIMENTAL

Data presented herein is reported as means±standard deviation values(error bars) unless otherwise indicated. A paired or unpaired Student's‘t’ test was used for statistical comparison between any 2 given samplesunless otherwise indicated.

Example I Emulsification of Regulatory T-Cell Modulating Agents

Preparing emulsions of water soluble factors using biodegradablepolymers is a commonly used strategy to deliver materials over extendedperiods of time and can make administration of factors with short halflives in vivo a reality. The use of thedouble-emulsion/solvent-evaporation technique is a commonly usedstrategy. Odonnell et al., Advanced Drug Delivery Reviews 28: 25-42(1997), See, FIG. 5. A frequently used polymer in these formulations ispoly-lactic-co-glycolic acid (PLGA), which is both biocompatible and FDAapproved. PLGA is also attractive because its degradation rates can becontrolled by the ratio of its monomers (lactide which is morehydrophobic, and glycolide which is more hydrophilic). This control overdegradation behavior provides the ability to release factors overperiods from days to months. Hanes et al., Pharm Biotechnol 6:389-412(1995). Microparticles prepared in this manner can carry large payloadsand encapsulate multiple agents including, but not limited to: i)cytokines (Thomas et al., J Pharm Sci 93: 1100-1109 (2004)); ii) smallpeptides (Haining et al., J Immunol 173:2578-2585 (2004)); iii) smallmolecule drugs; and iv) nucleic acids (Little et al., Proc Nad Acad SciUSA 101: 9534-9539 (2004)). The size can be controlled by parameterssuch as the concentration of the polymer solution, agitation speedsduring fabrication, and amount of surfactant used in the outer aqueousphase during the emulsification procedure. These formulations can beadministered in a variety of ways, including injection through a needleand syringe.

Any number of compounds may be emulsified using the above methodology.However, of most interest to the embodiments discussed herein, factorswhich are thought to influence the anti-inflammatory environment duringtolerance induction are completely compatible with the system describedhere (i.e., for example, IL2, TGF-β, and CCL22). Further, theemulsification and release of IL2 in PLGA microparticles using a doubleand single emulsion systems has recently been reported. Thomas et al., JPharm Sci 93: 1100-1109 (2004). Specifically, IL2 was encapsulated andreleased in an active state for periods of up to 4 months. Similarly,TGF-β has been encapsulated in microparticles using PLGA polymer blendswith release demonstrated for a period of over 1 month. Kempen et al., JBiomed Mater Res A 70:293-302 (2004); and Lu et al., J Biomed Mater Res50:440-51 (2000). CCL20, but not CCL22, has been encapsulated in PLGAmicroparticles which have been shown stimulate significant amounts of invitro dendritic cell chemotaxis (i.e., for example, over 0.5 mm fromtheir original position to the point of contact with the particle). Zhaoet al., Biomaterials 26:5048-5063 (2005) Importantly, this chemotaxiswas greater than that following bolus administration of the chemokine.CCL22 has been reported to induce regulatory T-cell chemotacticmigration both in vitro and in vivo. Curiel et al., Nat Med 10:942-949(2004).

Example II Labeling Artificial Presenting Cell Surface PolymerFunctionalization

This example provides a simple, rapid detection method to detectparticle surface polymer modifications capable of attaching to solubleand membrane T_(reg) stimulation factors.

Particle surface labeling of biodegradable polymers has beenaccomplished using several established techniques. The most commonstrategy for PLGA is to utilize carboxylic acid groups, which arepresent at the end groups and are generated upon degradation of theester bonds in its backbone. This can be accomplished using carbodiimidechemistry and can be facilitated using NHS esters. See, FIG. 6.

Another approach has been to create more of these carboxylic acid groupson the surface using a surfactant which is rich in these chemical groupsduring fabrication of the particle. Keegan et al., Macromolecules37:9779-9784 (2004). Also, short treatments with 0.1M NaOH can generatean abundance of these groups on the particle surface.

A new technique is presented in this example that quantifies surfacelabeling using a modification of the carbodiimide chemistry approach. Inthis technique, 2-pyridyldithioethylamine, reacts with activated estersand also contains a disulfide bond which is, in turn, readily reactedwith thiol compounds. See, FIG. 7 middle panel. This reaction liberatesa new chemical entity which absorbs light at 342 nm. See, FIG. 7 rightpanel.

Light absorption can be readily detected using any standard absorbanceplate reader. The absorbance data, along with the total amount ofparticles and their average surface area, can determine an estimate ofthe number of labeling events per unit area on the particle surface.This technique has successfully labeled the surface of 10 micrometerPLGA microparticles wherein dithiothreitol was used to liberate theabsorbent product (data not shown).

Alternatively, streptavidin may be used to facilitate the binding ofcommercially available biotin-labeled antibodies. For example,EDC/carbodiimide and NHS chemistry can take place in MES buffer in whichmicroparticles are suspended. The reaction tube will need to beconstantly rotated to keep particles evenly suspended for approximately2 hours. Particles will then be washed and 2-pyridyldithioethylamine(PDA) will be added to the activated ester by 4 hour incubation underagitation.

After washing, these particles are labeled with thiolated streptavidinin excess. Particles will be spun down and supernatants removed foranalysis at 343 nm.

Values obtained will be compared to a standard curve to determine thenumber of moles reacted/weight of particles. The average total availablesurface area for reaction can be determined by preparing an aqueoussuspension of particles with a known concentration. This suspension willbe analyzed by Coulter Counter to determine the number of particles perunit weight. The value, along with the average diameter of the particlesand the number of moles of streptavidin reacted/weight particles leadsto the number of streptavidin linkages per unit area on themicroparticle surface. This value can be checked with the use ofcommercially available biotin conjugated to a fluorescent linker.Particles could be labeled with this material, washed, dissolved usingNaOH/SDS, and this solution's fluorescence could be measured. Thisintensity, as compared to a standard curve, would indicate the amount oflabeling/weight of particles. A pH insensitive fluorescent probe wouldneed to be used in these experiments. These fluorescently labeledparticles will also be incubated (n=3) in a similar fashion as describedabove for the controlled release assay. Stability of the surface linkagewill be determined by the amount of fluorescence in the supernatantafter centrifugation (label that has been liberated from the particlesurface)

Example III Emulsification of Soluble T_(reg) Cell Factors in PLGAMicroparticles

This example describes the emulsification (i.e., for example,encapsulation) of soluble factors within PLGA microparticles using amodified double emulsion procedure. Odonnell et al., Advanced DrugDelivery Reviews 28:25-42 (1997).

Briefly, 20 μg of protein factor and 20 mg of bovine serum albumen willbe dissolved in 200 μL of sterile PBS and added to a solution of 100 mgof 10, 18-30, or 40-78 kDa PLGA with a lactide:glycolide monomer ratioof 50:50 in 2 ml of dichloromethane. This two phase system will besonicated for 10 seconds using a probe sonicator to form the primaryemulsion. The emulsion will immediately be transferred to 50 mL of ahomogenizing (5000 rpm), aqueous solution containing 5% by weightpoly(vinyl) alcohol (PVA) which serves as a surfactant. After 30 secondsof homogenization, the double emulsion is transferred to a stirringsolution of 1% PVA (100 mL) to allow for evaporation of thedichloromethane over a period of 3 hours. The particles will then becentrifuged at <150 rcf in a refrigerated centrifuge and the supernatantcontaining PVA will be decanted. Particles will be resuspended, washedwith cold water, and the process repeated 3×. Washed particles will thenbe suspended in a minimal amount of water and lyophilized for 3 days toallow sufficient time for removal of residual chloroform. Smallerparticles can be prepared using homogenization at 7000 rpm (1 μm).Larger particles can be prepared using a lower concentration ofsurfactant in the outer aqueous phase of the emulsions (i.e. PVA=1% and0.5% by weight) which will result in 10-20 μm particles.

Particle size will be determined by a Coulter Counter using volumeimpedance. Particle loading will be checked by exposing 5 mg ofparticles to a 0.5 M NaOH, 2% SDS solution to promote total degradationof the particles and dissolution of the protein. Concentrations can bedetermined using a BCA assay kit via the manufacturer's instructions andloading can be determined (n=3). It should be reasonable to assume thatthe relative amount of T_(reg) cell factor will remain the same withrespect to BSA during encapsulation. However, this assumption can bechecked using factor-specific ELISA in place of the BCA assay initially.A typical encapsulation efficiency using thedouble-emulsion/solvent-evaporation method for proteins is approximately50%.

Example IV Controlled Release of Soluble Factors from PLGAMicroparticles

This example provides a method to determine the controlled release ofsoluble factors from microparticles made in accordance with Example III.

Approximately 10 mg of microparticles (n=3) will be weighed out intomicrocentrifuge tubes and suspended in 500 μL of PBS. Tubes will besealed and placed on a shaker plate at 37° C. At 12 hour time intervals,tubes will be centrifuged, supernatants removed and stored at −70° C.,and new buffer will be added to the particles. At the end of 2 weeks,supernatants will be analyzed for T_(reg) cell stimulating factor usinga specific ELISA and release will be plotted over time with standarddeviations. Values obtained for all particle formulations in thesestudies can be used for setting up the appropriate conditions formeasuring the activity of these factors and determining their effects onT_(reg) cells in vitro.

Example V Primary Regulatory T-Cell Isolation

Lymph nodes will be harvested from BALB/c mice and processed to achievea single cell suspension. Magnetic cell sorting (MACS) will be employedto purify the population via a depletion step which enriches the CD4+population of cells (mostly CD4+CD25−) and a positive selection stepinvolving CD25 (resulting in mostly CD4+CD25+). Cells will be culturedthereafter in RPMI 1640 media including 10% FCS, glutamine, HEPES,nonessential amino acids, penicillin/streptomycin, and2-mercaptoethanol.

Example VI Biological Activity Of Encapsulated TGF-β and IL2

For the determination of TGF-β and IL2 activity, the effect of releasedfactors are measured on a population of CD4+ cells prior to positiveselection of CD25. This assay is takes advantage of the fact that TGF-βand IL2 can aid in the differentiation of peripheral CD4+ cells toincrease their expression of FoxP3 and suppress alloreactivelymphocytes. Fu et al., Am J Transplant 4:1614-27 (2004). Both TGF-β andIL2 are present at the same time as TCR engagement thereby facilitatingactivation.

CD4+ CD25− cells are treated for 5 days with experimental groups, mAbspecific to CD3 (1 μg/ml), and syngenic, lymphocyte-depleted andirradiated splenocytes. To examine biological activity of encapsulates,the following experimental groups (n=3) are used: 1) fresh, TGF-β andIL2 (1 ng/ml) as a positive control, 2) particles with no encapsulatedfactors and no external addition of IL2 or TGF-β, 3) IL2 particleincubation with the cells+(1 ng/ml fresh TGF-β), 4) TGF-β particleincubation with the cells+(1 ng/ml fresh IL2).

After treatment, cells will be analyzed by flow cytometry for theincreased presence of CD25 and intracellular expression of FoxP3 (viaCytofix/Cytopenn kit and labeled FoxP3 mAb) which would indicateactivation of the cells. The amount of particles to be used in theseexperiments will be based on the results of the controlled release assay(described above) to allow for equivalent amounts of administered growthfactor. Groups 3 and 4 will be compared to Group 1 to determine thebiological activity of the released IL2 and TGF-β, respectively.

Example VII Measurement of T_(reg) Cell In Vitro Chemotaxis

This example provides one method for measuring the in vitro chemotaxisof T_(reg) cells in response to chemokine release (i.e., for example,CCL22) from particles. See, FIG. 8.

Regulatory T-cells will be isolated as described above using greenfluorescent protein transgenic mice (GFP+−C57BL/6 mice). This providesfluorescently labeled cells which have not been treated by any externalreagents. Microparticles containing CCL22 (n=3) will be placed belowopaque, transwell filters (mean pore size=3 μm and 5 μm). Cells will beseeded on top of this membrane and allowed to incubate for 5 hoursbefore measuring fluorescence. A top and bottom fluorescent readingformat is used which allows for the determination of the number offluorescent cells that have migrated through the membrane at any point.No checkerboard analysis is needed to differentiate between chemotaxisand chemokinesis given that the only factor we are using in ourexperiments is a well-known chemotactic agent.

Alternatively, a carboxyfluorescein diacetate succinimidyl ester, orCSFE (a factor used to track T-cell proliferation), may be used insteadof the GFP transgenic mice to label the T_(reg) cells. A manual countingof cells both below and above the transwell filter (using unlabeledcells as a control) should be performed to determine if CSFE has anyeffect on the chemotaxis of the regulatory T-cells.

Experimental controls include, but are not limited to: 1) wells withblank microparticles (as a negative control); and 2) wells containingsoluble CCL22 at 0.5-20 ng/ml (the typical manufacturer's stated ED50range for this material) as a positive control. The amount ofmicroparticles used in this experiment are derived from the controlledrelease experiment (described above) to equalize the released CCL22 withthe positive controls.

A t-test using two-tailed, unequal variance will be used to determinedifferences between groups with p<0.05 being deemed significant.

Example VIII In Vitro Measurement Of Soluble Released Factors

This example identifies one embodiment for measuring the effect ofsurface co-stimulatory and soluble released factors on T^(reg) cells invitro.

Materials

Latex beads with streptavidin can be easily purchased in 1, 5, and 10 μmsizes and can be labeled in one step with biotinylated mAb specific toCD3 and CD28. Optionally, the surface bound moieties can be optimized

In addition to determining the optimal size for the surface labeledmicroparticles, label ratios are also varied to optimize theproliferation of primary T_(reg) cells (i.e., for example, 0% CD3/100%CD28; 25% CD3/75% CD28, 50% CD3/:50% CD28; 75% CD3/25% CD28: 100% CD3/0%CD28).

Furthermore, saturating concentration of the stimulatory factors on theparticle surface may or may not be optimal for T_(reg) cellproliferation. For example, stimulation of regulatory T-cells by CD28requires an optimized dose. Fu et al., Am J Transplant 4:1614-27 (2004);Salomon et al., Immunity 12:431-40 (2000). To determine this dose, thetotal percentage of streptavidin sites accessible to mAb bound biotinare reduced (i.e., for example, by limiting the addition of free biotinduring the labeling procedure). All samples (n=3) will be compared usinga two-tailed t-test (unequal variance) with p<0.05 being deemedsignificant.

Methods

Primary T_(reg) cells will be isolated from C57BL/6 mice and expandedusing helper cell-free in vitro conditions including the addition offresh IL2. The latex bead system will serve as a positive control forcomparison with labeled PLGA microparticles of different sizes. Negativecontrols will include untreated cells and cells treated with particleswithout surface label.

Soluble IL2 will be replenished, and the cells recounted bi-weekly,while new particles will be administered twice over a two week period.Besides measurement of total regulatory T-cell proliferation, sampleswill be taken from the culture flasks bi-weekly for flow cytometryanalysis. Cells will be stained with fluorescently labeled mAbs whichrecognize CD25, and CD4. Intracellular staining for FoxP3 is used todetermine the activation state of the regulatory T-cells.

Labeling both IL2 and IL2+TGF-β encapsulated particles are performedunder the same conditions which produced the optimal surface labeledformulation. Also, this protocol will allow the determination if anartificial antigen presenting cell is capable of activating and inducingthe proliferation of regulatory T-cells without the need for manualaddition of soluble factors.

Example IX In Vivo Injection of Chemokine Encapsulated Particles

This example demonstrates the injection of chemokine encapsulatedparticles to determine recruitment capacity in vivo.

Particles will be prepared with the full amount of CCL22 (as describedin the first experimental section), ⅓ this amount, and 1/10 of thisamount. Primary cells (1×10⁵) from transgenic GFP+−B6 mice will beadoptively transferred into normal B6 mice for in vivo chemotaxisexperiments. At this time mice will be given an injection of the threetypes of CCL22 encapsulated microparticles in a solution of sterile PBSand 1% carboxymethylcellulose (to avoid particle settling and needleclogging with the high concentrations of particles used). This injectionwill either be on a shaved and sterilized belly of the animal (i.e., forexample, intraperitoneally) (n=3) or in the tibealis anterior muscle(hind leg) (n=3). On the other side of the animal, blank microparticleswill be injected S.C. or I.M. as a control. Another experimental groupprovides an S.C. or I.M. injection to mice using a soluble CCL22 in thesame amount as the CCL22 encapsulated particles (full amountencapsulated) (n=3).

At t=1 day and 3 days, mice will be euthanized and skin or muscle at thesite of an injection will be resected for histology sections. In thesesections, the more GFP expressing cells near the site of the depot, thegreater the success of the formulation. For a more quantitativeanalysis, we will form a single cell suspension with the resected tissueand analyze using flow cytometry (at 488 nm) to determine how manyadoptively transferred regulatory T-cells were recruited compared to theblank microparticle-treated, negative control (deemed statisticallysignificant by t-test, p<0.05).

Example X In Vivo Injection of Artificial Antigen Presenting Cells

This example demonstrates the injection of artificial antigen presentingcells to determine manipulative effect on T_(reg) cells in vivo.

Artificial antigen presenting cells will be prepared by encapsulating anoptimized CCL22 formulation plus IL2 and TGF-β followed by an optimizedsurface labeling strategy. Experimental groups will be designed toexplore the necessity of the included factors for stimulation of therecruited regulatory T-cells in vivo (i.e. for example, one formulationwith each soluble and surface stimulating component being absent). Micewill be adoptively transferred with transgenic, GFP+ regulatory T-cellsas described above and administered injections in the same manner, butthis time with the artificial APC formulations instead of encapsulatedCCL22 alone (n=3 for each group). Cells from resected skin and musclewill be intracellularly stained for the level of FoxP3 expression (giventhe strong correlation between its up-regulation and T_(reg) cellstimulatory capacity).

These experiments are repeated with the intent of sorting CD4+ cellsinstead of intracellular FoxP3 staining. The sorted cells willsubsequently be used as suppressor cells (suppressor: responder ratiosof 1:1, 2:1 and 5:1) with freshly isolated CD4+CD25− which have beenpre-labeled with CSFE (15 μM) and stimulated for 3 days using monoclonalantibodies specific for CD3 (1 μg/ml) and syngenic, irradiatedsplenocytes (depleted of lymphocytes). Cultures will be monitored usingflow cytometry. Efficient suppression would result in a decreaseddilution (or higher signal at 488 nm) of CSFE in the cellular populationdue to the fewer cell divisions of a suppressed lymphocyte. To determinethe necessity of each of the individual factors, experimental groupswill be compared via two tailed t-test assuming unequal variance withp<0.05 being considered significant.

Example XI Induction of Allograft Tolerance Using Artificial AntigenPresenting Cells

This example demonstrates therapeutic use of optimized artificialantigen presenting cells for induction of allograft tolerance.

The results of the in vivo suppressor assay (supra) provides optimizedformulations and are used for the in vivo allograft tolerance assays.These optimized formulations will be co-administered using either adepot administration of, or by coating the transplant in, artificialantigen presenting cells (n=5). Controls will include mice with notreatment and mice administered (15 mg/kg day) cyclosporine S.C. as apositive control (n=5). Furthermore, an optional experimental groupcomprising an artificial APC group with an initial cyclosporine dose of15 mg/kg day, and then reduced to examine the effect ofimmunosuppressant promotion or inhibition of immune tolerance induction(n=5). The experimental endpoint will be 100 days post operation.

For skin transplantation, mice will be shaven, sterilized, andanesthetized before preparation of two graft beds on the posterior chestwall. Rosenberg A. S. (1991), eds. Coligan, J. E., Kruisbeek, A. M.,Margulies, D. H., Shevach, E. M. & Strober, W. (John Wiley and Sons, NewYork), pp. 4.4.1-4.4.12. These graft beds will engraft full thicknessskin grafts from the tail of a donor and syngenic control. After oneweek of recovery, the transplant will be monitored daily for redness,hair growth, hemorrhaging, and status of graft borders. The percentageof viable skin will be recorded daily until only 10% remains, at whichtime the animal will be euthanized.

Heterotopic heart transplants will be performed as previously described.Zhang et al., Transplantation 62:1267-1272 (1996). Rejection will bemonitored via direct abdominal palpitation using the following rankingsystem: A). Strongly beating, B) Noticeable decline from A, or C)Termination of pulsation at which time the animal will be euthanized.

Following termination of the animals in both transplant models, theallografts will be fixed in 10% formaldehyde and sent for embedding,staining, and evaluation of rejection. Percentage of surviving grafts ineach group will be plotted against time for comparison.

A log rank test will be used to compare groups with transplantationrejection data as analyzed by any one of many available softwarepackages.

Example XII Restoring Immunological Regulation in Periodontal DiseasedTissues Results Overview

In this experiment, artificial antigen presenting cells providing aCCL-22 controlled release formulation were injected into palatalgingival tissue of right molars of mice (n=3) at the times of −1, 10 and20 days of periodontal infection. At 30 days, total RNA was extractedfrom periodontal tissues as previously described and analyzed forregulatory T-cell and periodontal disease factor. Garlet et al., “Theessential role of IFN-gamma in the control of lethal Aggregatibacteractinomycetemcomitans infection in mice. In: Microbes andinfection/Institut Pasteur (2008). Infected, untreated mice (AA) andnon-loaded particle treated (CP) groups had low levels of FoxP3 (aregulatory T-cell marker) and IL10 (a suppressive cytokine secreted byT_(reg) cells), but elevated levels of RANK-L (an osteoclastdifferentiation factor). See, FIGS. 12A & 12B. Conversely, CCL-22particle treatment groups (LP) had significantly higher levels of FoxP3and IL10, and significantly lower levels of RANK-L. To determine if theparticles had therapeutic effect, mice were given the aforementionedtreatments and alveolar bone loss was measured at 30 days as described.Garlet et al., “Actinobacillus actinomycetemcomitans-induced periodontaldisease in mice: patterns of cytokine, chemokine, and chemokine receptorexpression and leukocyte migration”. Microbes and infection/InstitutPasteur 7, 738-747 (2005). Mice treated with CCL-22 loaded particles(LP) exhibited significantly reduced bone loss compared to neg.controls. See, FIG. 12B.

Methods

Fabrication and Characterization of Controlled Release Formulations:Encapsulation of recombinant murine CCL-22 into PLGA microparticles wasperformed using a double emulsion procedure to generate particles in the10 μm range (supra). Loading was verified by dissolution (NaOH, DMSO)followed by protein detection assays such as ELISA to detect totalprotein encapsulated. Particle sizing and SEM analysis were performedfor quality control purposes.

Example XIII Therapeutic CCL-22 aAPCs in a Murine Mouse Model forPeriodontal Disease

To induce periodontal disease in a mouse, we delivered 1×10⁹ CFU of adiluted culture of Aggregatibacter (Actinobacillus)actinomycetemcomitans JP2 (anaerobically grown in supplemented agarmedium, TSBV) in 100 μl of PBS with 2% carboxymethylcellulose, placed inthe oral cavity of mice with a micropipette in accordance with ExampleXII. This procedure will be repeated at 48 and 96 hours later to fullyestablish periodontal disease.

Experimental groups will comprise of eight-week-old C57B1/6 mice. CCL22loaded particles will be injected in the palatal gingival tissue ofright molars from the mesial of first molar until the distal face of thethird molar, at the times of −1, 10 and 20 days of infection. Negativecontrols will include non-infected and sham-infected mice, which receiveheat-killed bacteria in 2% carboxymethylcellulose solution, mice thatreceive PBS injection in left maxilla of the same LP group, and micethat receive the injection of control non-loaded particles. For apositive control, we will utilize mice that receive regular IPinjections of VIP (vasoactive intestinal peptide) as this materialinduces systemic proliferation of regulatory T-cells. Chorny et al.,“Vasoactive intestinal peptide induces regulatory dendritic cells thatprevent acute graft-versus-host disease while maintaining the graftversus-tumor response” Blood 107:3787-3794 (2006). After 30 days ofinfection, mice will be euthanized and the samples collected for thefollowing experimental analyses:

Alveolar Bone Loss/Healing Analysis. Evaluation of the extent ofalveolar bone loss will be performed in accordance with Example XII andFIG. 12 above. Further, quantitative histological methodology will beused to characterize bone healing. Briefly, the maxillae will behemisected, exposed overnight in 3% hydrogen peroxide and mechanicallydefleshed. The palatal faces of the molars will be photographed at 20×magnification using a dissecting microscope (Leica, Wetzlar, Germany),and the images will be analyzed using ImageTool 2.0 software (Universityof Texas Health Science Center, San Antonio). Quantitative analysis willbe used for the measurement of the area between the cement-enameljunction (CEJ) and the alveolar bone crest (ABC) in the 3 posteriorteeth, in arbitrary units of area (AUA). In total, 5 animals will beanalyzed, and for each animal, the alveolar bone loss will be defined asthe average of CEJ-ABC between the right and left arches.

Histological Assessment for Bone Healing. At the specified times,animals will be euthanized and block sections removed forimmunohistochemistry analyses. Tissue sections will be divided into 2groups (every other section), one for histological/histomorphometryanalysis and the other for immunohistochemistry to assess new boneformation by staining for the Dentin Matrix Protein 1 and BoneSialoprotein.

Real-Time PCR Analysis. Extraction of total RNA from periodontal tissues(upper molars with whole surrounding buccal and palatal periodontaltissues) will be performed with Trizol reagent (Invitrogen, Rockville,Md.) and cDNA synthesis. RealTime-PCR quantitative mRNA analyses will bedone in a MiniOpticon system (BioRad, Hercules, Calif.), using SybrGreenMaster Mix (Invitrogen), 100 nM specific primers, and 2.5 ng of cDNA ineach reaction. We will analyze tissues for the presence of CCL-22,FoxP3, IL-10, RANK-L, and TGF-b. For mRNA analysis, the relative levelof gene expression is calculated in reference to beta-actin using thecycle threshold (Ct) method.

Quantification and Visualization of Regulatory T-cells. Tissue will beresected and either: 1) sectioned for staining specific for regulatoryT-cells using reported methods (Raimondi et al J Immunol 176, 2808-16(2006) or 2) digested and separated using Ficoll in order to isolatewhite blood cells for flow cytometric analysis. The latter will allow usto determine the degree of FoxP3 expression specifically in regulatoryT-cells (directly correlating to suppressive activity and also thepercentage total regulatory T-cells in the periodontal space incomparison to other lymphocyte populations, CD4+CD25−, CD8+.

Statistical analysis. Preliminary data (n=3 mice per group with aninternal control) obtained statistical significance which was slightlyless than the p=0.05 cutoff. Power analysis based upon standarddeviations and response magnitudes in pilot data (assuming power=0.8)indicates that we require n=5 to obtain desired confidence in ourexperimental results. Statistical significance between the infected andcontrol mice of both strains will be analyzed by ANOVA, followed byBonferroni post test. Values of P<0.05 will be considered statisticallysignificant. We will also test both 1 month and 2 month time-points toexamine whether or not therapeutic efficacy can be maintained andwhether or not healing occurs at these time-points. PCR and AUA basedmeasurements of bone loss/healing can be performed on the same set ofmice (n=5×2 time points×4 groups) and histological analysis and flowcytometry can be performed on another set of mice (n=5×2 time-points×4groups) leading to a total of 80 mice. Experimental groups include: 1)sham control, 2) blank particle control, 3) VIP positive control, and 4)CCL-22 particle experimental group.

Example XIV Making Anisotropic Microspheres with Soft Protein Islets

Commercially available carboxylated phosphatidylserine (PS) microspheres(mean diameter 6.37 μm) were purchased from Bangs Laboratories Inc, USA.In preparation for the procedure, microspheres were washed twice indeionised water, then washed once in ethanol, centrifuged at 3500 rpmfor 5 min, and re-suspended in water. Microbiology-grade glass coverslips were washed in soap solution, distilled water, and then ethanol(70%) under bath sonication for 15 min each. “Microwells” were developedby spotting 3 μl of microsphere suspension (10% w/v in water) onto acover slip and dried sequentially at 25° C. for 1 hr 40° C. for 30 minand 60° C. for 10 min (Tg of PS bulk is 100° C. and reduces to 70° C. asthe thickness reduces) 27 to avoid surface melting. The wells werefurther filled 4 times with concentrated microsphere suspension (30%w/v) in water with intermittent drying at 4-8° C. between additions. Thecolloidal crystals were further dried at room temperature for 3 hours.Prior to the addition of PDMS, cover slips with colloidal crystals werepreheated to 60° C. for 5 min on a leveled hot plate (The PDMS((PDMS/catalyst) (10:1) (w/w)) (Sylgard 184 silicone elastomer kit,Dowcorning Corp, MI, USA) solution was added and allowed to saturate andstabilize the interstitial spaces for a minute. This mixture was thenimmediately heated to 90° C. for 15 min. After setting of the PDMSscaffold (16 hrs at 40° C.), the cover slip was carefully removed toprepare the PDMS scaffold/colloidal crystal for protein patterning.

For protein labeling, scaffolds were first immersed in 0.1% Tweensolution, to reduce the non-specific adsorption of proteins. Biotin-PEO(EZ Link®, Pierce, USA) was immobilized onto the microspheres in thePDMS scaffold. Carboxylate groups on the surface of microspheres (exceptthe region at PDMS mask) were activated with 0.1M1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (Acros, N.J., USA),and 0.2M N-hydroxysulfosuccinimide (Sulfo-NHS) (Thermo scientific, IL,USA) in 2000 in 0.2M MES (pH5.5) buffer for 2 hours. Following thereaction, microspheres were washed two times with MES buffer prior toaddition of 50 mM biotin-PEO (Fisher scientific, USA) in 200 μl MESbuffer. After 2 hrs, the solution was removed and microspheres werewashed two times with MES buffer in the scaffold. For etching, TetraButyl Ammonium Flouride (TBAF) (1M solution in Tetrahydrofuran,sigma-Aldrich Inc) in 1-Methyl, 2-Pyrrolidininone (NMP) (Sigma-AldrichInc)/Deionised Water (1:6) (v/v) in 0.1% Tween 80 solution was used.After 1 hour incubation of microspheres in etchant solution, thescaffolds were washed two times with the MES buffer. The second proteinwas then immobilized to the newly exposed area using EDC-NHS chemistry.For that the succinimide derivatized particles 200 μl of 0.2 mM albuminrhodamine (Albumin from bovine serum, tetramethyl rhodamine conjugate,Invitrogen) in 0.1% Tween 80/MES buffer (pH 5.5) was added to label thepatches. The reaction proceeded for 2 hrs in the dark followed bywashing with 1% (w/v) Bovine Serum albumin (BSA) (Sigma-Aldrich Inc) in200 μl MES (pH 5.5) buffer. For labeling of the remaining surface of theparticle, 200 μl MES buffer (pH 5.5) with 0.1% Tween and 1% BSAcontaining 0.2 mM avidin flourescein (Immunopure®, Pierce, USA) wasadded to the washed microspheres in the scaffolds. After 2 hours, thescaffolds were washed two times with MES buffer (pH5.5), and furtherwashed one time in deionised water prior to storage at 4-8° C. Themicrospheres were scrapped out of the scaffold with a steel spatula.Optical microscopy images were taken with an Optical microscope (Caltexsystems 3D Digital Video Inspection measurement system+signatone 1160Probe Station). SEM studies were done with JSM 6330F. CLSM studies wereperformed using an Olympus FluoroView 1000 confocal microscope.

Example XV Microparticle Fabrication

Rationally design of controlled release microparticles may be createdusing a predictive mathematical model. S. R. Little, S, N. Rothstein, W.J. Federspiel, Journal of Materials Chemistry 18, 1873 (2008); S, N.Rothstein, Federspiel, W. J., Little, S. R., Biomaterials, (2009). Themodel allows computationally design formulations with specific releaseprofiles based on drug/polymer characteristics and the desired releasekinetics. Computer generated fabrication parameters are then used toinform the production process. Specifically, emulsion technique canencapsulate drugs in microparticles. FIG. 21. This model specifies thatformulations should have the following characteristics to produce a 20day linear release profile: Particle Size=20 μm; Internal Drug PocketSize=500 nm; Average Polymer Molecular Weight=15 kDa;Polydispersity=1.5. FIG. 16.

Furthermore, a VIP (vasoactive intestinal peptide) microparticleformulation will be designed to provide a constant supply of the peptideover 20 days using the following characteristics: Particle Size=20 μm;Internal Drug Pocket Size=300 nm; Average Polymer Molecular Weight=15kDa; Polydispersity=1.5. If VIP release behavior deviates from thedesigned profile, we are able to easily reformulate the microparticlesto appropriately address the deviant characteristic. Release of VIP frommicroparticles are expected to provide data similar to FIG. 16.

Example XVI Preparation of iTreg Cells

This example describes one method of preparing iTreg cells following anin vitro administration of soluble vasoactive intestinal peptide.

Animals

Six-eight week-old C57BL/6 and B6.SJL-Ptprca/BoyAiTac (CD45.1) mice werepurchased from Taconic and used within two months of delivery.B6(Cg)-Tyrc-2J/J (albino C57BL/6 mice) were purchased from The JacksonLaboratory. C57BL/6.Luc+ mice were a kind gift from Dr. Stephen Thorne(Dept. of Surgery, University of Pittsburgh). All animals weremaintained under specific pathogen-free conditions.

Materials

Mouse CD4 negative isolation kit, αCD3/αCD28-labeled beads (aAPC Dynal®)and Vybrant CFDA-SE cell tracer kit were from Invitrogen Corporation(Carlsbad, Calif., USA). Recombinant mouse IL-2 (R&D systems,Minneapolis, Minn., USA), and vasoactive intestinal peptide.

T Cell Isolation

Spleen and lymph nodes are dissected from mice, and single cellsuspensions are prepared using mechanical digestion. Following RBClysis, CD4+ cell isolation are performed using the CD4 negativeisolation kit (Invitrogen) as per the manufacturer's instructions. Toenrich for CD25− cells, CD4+ cells are incubated with anti-mouse CD25−PE antibody (eBioscience) followed by addition of anti-PE microbeads(Miltenyi). Bead-bound CD25+ cells are isolated by passing cells througha magnetic column. Unbound CD4+ CD25− cells are separated and used toinduce Treg.

Treg Induction

Freshly-isolated naïve CD4+ CD25− cells are cultured with aAPC Dynal®beads at a 2:1 (dynal:cell) ratio in the presence of betweenapproximately 3-50 ng/ml vasoactive intestinal peptide. To obtaineffector T cells (Teff), CD4+ CD25− cells are cultured with aAPC Dynal®beads and 10 ng/ml IL-2 only. Cell cultures are maintained for 4 days,and cells separated subsequently from the magnetic Dynal® beads. Todetermine induction of Treg phenotype, FoxP3 staining and flow cytometry(BD-LSRII) are performed at the end of the 4-day culture period as perthe manufacturer's instructions (eBioscience).

Example XVII In Vitro Suppression Assay

Freshly-isolated naïve CD4+ CD45.1+ cells are stained withCarboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen, asper the manufacturer's instructions) and co-cultured with autologousinduced Treg (generated as described above) at different ratios in96-well plates. The number of naïve CD4+ CD45.1+ cells is always 50,000cells/well. For stimulation, 25,000 aAPC Dynal® beads per well are used(2:1, naïve cell:Dynal ratio). Co-cultures are maintained for 4 days,followed by staining for flow cytometry.

Example XVIII In Vitro iTreg Stability Testing

iTreg cells generated under different conditions and effector T cells(Teff, generated by stimulating naïve T cells in the presence of IL-2only) are obtained from 4-day cultures and rested in 10 ng/ml IL-2 for 2days. Thereafter the cells are cultured along with aAPC Dynal® beads asstimulators: (i) for Dynal® only (no Teff and no factors) group, 100,000iTreg were cultured with 200,000 Dynal® beads along with 10 ng/ml VIP;(ii) for Dynal®+Teff (no factors) group, 50,000 iTreg cells are culturedwith 50,000 Teff cells and 100,000 Dynal® beads along with 10 ng/ml VIP;(iii) for Dynal®+factors (no Teff) group, 100,000 iTreg cells arecultured with 200,000 Dynal® beads and respective factors at theconcentrations described above; (iv) for Dynal®+Teff+factors group,50,000 iTreg cells are cultured with 50,000 Teff cells and 100,000Dynal® beads and respective factors. Re-stimulation experiments werecarried out for 4 days and cells were then stained and analyzed by flowcytometry.

Example IXX VIPMP Formulations

VIP microparticles (VIPMPs) were prepared following calculations using amodel guided fabrication protocol. Rothstein et al., “A simple modelframework for the prediction of controlled release from bulk erodingpolymer matrices” J of Mater. Chem. (2008) 18:1873-1880. Themathematical algorithm of the computer model suggests specific polymerratios to achieve desired release kinetic profiles. Separate batches ofmicroparticles were fabricated which could then be combined inmodel-specified ratios to obtain complex release behaviors.

Three batches were comprised of individual PLGA polymer molecularweights using 4.2 kDa polymer, RG502H PLGA polymer (12.6 kDa), and RG505polymer (55 kDa). Additionally, a fourth batch was fabricated with the12.6 kDa polymer which contained poly(ethylene glycol) (PEG) atapproximately 4×10⁴ mM in the inner aqueous phase. The inner aqueousphases of all VIPMPs comprises approximately 1250 μg/ml VIP. Unloaded(or “blank”) sets of particles were also fabricated for each batch.

In a separate study, nonporous and porous (with an inner aqueous phasecomprised of 7.5 mm NaCl) VIP microparticles were prepared using RG502Hpolymer (approximately 12.6 kDa). The inner aqueous phases of thenonporous and porous microparticles consisted of 1250 μg/ml VIP. Blanksets of nonporous and porous particles were also fabricated.

Example XX VIPMP Release Assays

Release samples were collected for each of the four batches of VIPMPsmade in accordance with Example XI. VIP release was also measured fortwo sets of VIPMPs with varying polymer ratios determined by a computermodel that predicted a linear and multi-bolus release profile,respectively.

For the predicted linear release profile VIPMPs, the computer modelsuggested a microparticle composition comprising ratios of 10.6% of the4.2 kDa polymer, 31.9% of the 12.6 kDa polymer (without PEG), and 57.5%of the 100 kDa polymer.

For multi-bolus release profile VIPMPs the computer model suggested amicroparticle composition comprising ratios of 33.3% each of the 4.2kDa, 12.6 kDa (with PEG), and 100 kDa polymers.

Release was also measured for the porous and nonporous microparticlegroups. Additionally, eight sets of blank microparticles for each groupwere also collected at each time-point. Release assays in physiologicalbuffered saline (PBS) conducted for each set of microparticles. Tenmilligrams of blank microparticles or VIPMPs and 1 mL of either media orPBS were incubated at 37° C. Vials were centrifuged at 2000 rpm, and 800μL of supernatant was removed and saved at −80° C., and replaced withfresh PBS for each time point.

VIP concentration was determined using a VIP EIA kit purchased fromPhoenix Pharmaceuticals. Samples were diluted up to 10×.

Example XXI VIP Bioactivity Assays

Dendritic cells were isolated from murine bone marrow and culturedfollowing standard procedures.

In the first two experiments, both GM-CSF and IL-4 were added to DCmedia (modified RPMI 1640) of bone marrow cell cultures during mediachanges on Days 0, 3 and 6. In the third experiment, the IL-4 waspurposely omitted and GM-CSF was added in 5× concentration. On Day 6,CD11c+ DC cells were isolated using MACS cell sorting beads and platedin a 24-well plate at 100,000 cells/well. Five different groups of fourwells each were analyzed and received treatment. An immature DC controlgroup did not receive LP S. A mature DC control group received only LPS.Three different groups were matured with LPS and received differentconcentrations of either soluble VIP or releasates of VIP microparticlesor blank microparticles (using the 12.6 kDa polymer).

Preliminary Protocol: Soluble VIP was added to the third group inconcentration of 2.5×10⁻⁸ M. Ten milligrams of microparticles were firstincubated for 4-6 hours in media and 250 μL of releasates were added toeach well. After treatment, the DCs were incubated for approximately 18hours. Prior to the CD4+ T cell chemotaxis study, the 24-well plate wascentrifuge and the media was removed. The cells were then starved with600 μl, PBS+1% BSA and incubated for one hour.

Dose Response Protocol: Soluble VIP was added in concentrations of 10⁻⁶M, 10⁻⁸ M, and 10⁻⁹ M to both immature DC groups and DCs matured withLPS. Twelve different groups were matured with LPS and receiveddifferent concentrations of either blank porous, blank nonporous, VIPporous, or VIP nonporous microparticles (of the 12.6 kDa polymer).Microparticles were added in concentrations of either 2.77 mg/mL, 1.39mg/mL, and 0.277 mg/mL. In a second DC culture, the low concentration ofmicroparticles was reduced to 0.0556 mg/mL. At time-points of 7, 24, and48 hours, 100 μl was removed from each well and frozen for ELISAanalysis. 100 μl of fresh media was then added to each well after sampleremoval to maintain a total well volume of 600 μl.

For chemotaxis studies, in the first two experiments, both GM-CSF andIL-4 were added to DC media (modified RPMI 1640) of bone marrow cellcultures during media changes on Days 0, 3 and 6. In the thirdexperiment, the IL-4 was purposely omitted and GM-CSF was added in 5×concentration. On Day 6, CD11c+ DC cells were isolated using MACS cellsorting beads and plated in a 24-well plate at 100,000 cells/well. Fivedifferent groups of four wells each were analyzed and receivedtreatment. An immature DC control group did not receive LPS. A mature DCcontrol group received only LPS. Three different groups were maturedwith LPS and received different concentrations of either soluble VIP orreleasates of VIP microparticles or blank microparticles (using the 12.6kDa polymer). Soluble VIP was added to the third group in concentrationof 2.5×10⁻⁸ M. Ten milligrams of microparticles were first incubated for4-6 hours in media and 250 μL of releasates were added to each well.After treatment, the DCs were incubated for approximately 18 hours.Prior to the CD4+ T cell chemotaxis study, the 24-well plate wascentrifuged and the media was removed. The cells were then starved with600 μL PBS+1% BSA and incubated for one hour prior to chemotaxisstudies.

Example XXII Microparticle Characterization

The size distribution of each set of microparticles was determined usinga Coulter counter. Additionally, scanning electron microscopy was usedto analyze microparticles.

Example XIII ELISA Test for CCL22 Production

An ELISA test purchased from R&D Systems was used to measure CCL22production by dendritic cells following modified lab protocol. Analternate blocking buffer of 1 mL Tween dissolved in 50 mL PBS was used.Samples were diluted up to 500×.

Example IVXX CD4+ Cell Isolation and Chemotaxis

CD4+ T-cells were isolated from the spleen and lymph nodes of a mouseusing Dynal beads following the lab protocol. Transwells were added tothe 24-well plate containing the DCs and 100 μL of PBS and 1% BSAcontaining 500,000 CD4+ cells were added to each transwell and incubatedfor two hours. T Cells that flowed through were analyzed for FoxP3+expression using flow cytometry.

Example XXV Murine Periodontitis Model

Wild Type C57BL/6 mice were infected with Actinobacillusactinomycetemcomitans three times during the first week of a 30 day testto initiate the periodontal disease. A ‘sham’ control group received nobacteria or microparticles and an ‘untreated’ group received onlybacteria. A ‘blank’ control group will receive bacteria and injectionsof unloaded microparticles. All microparticles were administered at aconcentration of 1.25 mg/50 μL in sterile PBS with 2%carboxymethylcellulose. Microparticles were administered on days −1, 10and 20 into the periodontal pockets of right maxillary molars. VIPmicroparticles were injected into a fourth group of infected mice as apotential treatment. On Day 30, all mice were sacrificed and themaxillae were harvested for alveolar bone loss analysis.

Example XXVI Evaluation of Bone Loss

Mice maxillae were incubated in 1× dispase overnight at 37° C. and thenplaced in hydrogen peroxide for two hours to remove any soft tissue. Thearea between the cementoenamal junction (CEJ) and the alveolar bonecrest (ABC) on the buccal side of right maxilla was measured using adissecting microscope and CellSans software.

Example XXVII IL-4 Mediation Of Chemotaxis

IL-4 will be omitted from an in vitro chemotaxis study to verify thatVIP promotes tolerogenic phenotypes in DCs and not IL-4. It is expectedthat the omission of IL-4 will have no effect on chemotaxis. ELISAs willbe used to measure CCL22 production of DCs cultured with VIPmicroparticle releasate in the absence of IL-4.

Example XXVIII Induction of Naïve CD4+CD25+-Naïve T Cells

This example will determine if released VIP is able to directly inducenaïve CD4+CD25− naïve T cells to exhibit regulatory phenotypes as shownin literature. Fernandez-Martin et al., (2006) “Vasoactive intestinalpeptide induces regulatory T cells during experimental autoimmuneencephalomyelitis” Eur. J. Immunol. 36:318-326.

Example LXXX Repetitive Measures on In Vivo Murine Model PeriodontisisTreatment

This example will verify the above in vivo murine model experiments byincreasing the sample size and improve statistical significance.

QCPR analysis and flow cytometry will also be conducted to measure theexpression of FoxP3+ T cells (Tregs) in draining lymph nodes and in theperiodontal pocket. A higher expression of FoxP3+ T cells (Tregs) indraining lymph nodes is expected to show that VIP microparticles inducethe production of Tregs. Additionally, an increase in tolerogenic DCs indraining lymph nodes is expected and can be evaluated by observing adecrease in CD86 and CD80 and MHCII on the DCs using flow cytometry.

We claim:
 1. A method, comprising: a) providing; i) a patient comprisinga target tissue and a blood cell population, wherein said target tissueexhibits at least one symptom of a disease; and ii) a microparticlepopulation comprising a vasoactive intestinal peptide; b) administeringsaid microparticle populations to said patient under conditions suchthat said blood cell population is induced; and c) contacting saidinduced blood cell population with said target tissue such that said atleast one symptom of said disease is reduced.
 2. The method of claim 1,wherein said disease comprises an immunological disease.
 3. The methodof claim 1, wherein said blood cell is a white blood cell.
 4. The methodof claim 3, wherein said white blood cell is a T cell.
 5. The method ofclaim 4, wherein said T cell is a regulatory T cell.
 6. The method ofclaim 4, wherein said white blood cell is a B cell.
 7. The method ofclaim 1, wherein said microparticle population comprises a polymercomposition predicted by a mathematical algorithm.
 8. The method ofclaim 7, wherein said polymer composition comprises a 4.2 kDa polymer, a12.6 kDa polymer and a 55 kDa polymer.
 9. The method of claim 7, whereinsaid polymer composition comprises a 4.2 kDa polymer, a 12.6 kDa polymerand a 100 kDa polymer.
 10. A microparticle comprising a 4.2 kDa polymer,a 12.6 kDa polymer and a 100 kDa polymer and a vasoactive intestinalpeptide.
 11. The microparticle of claim 10, wherein said 4.2 kDa polymercomprises 10.6% of said microparticle.
 12. The microparticle of claim10, wherein said 12.6 kDa polymer comprises 31.9% of said microparticle.13. The microparticle of claim 10, wherein said 100 kDa polymercomprises 57.5% of said microparticle.
 14. The microparticle of claim10, wherein said microparticle further comprises polyethylene glycol.15. A microparticle comprising a 4.2 kDa polymer, a 12.6 kDa polymer anda 55 kDa polymer and a vasoactive intestinal peptide.
 16. Themicroparticle of claim 15, wherein said 4.2 kDa polymer comprises 33.3%of said microparticle.
 17. The microparticle of claim 15, wherein said12.6 kDa polymer comprises 33.3% of said microparticle.
 18. Themicroparticle of claim 15, wherein said 55 kDa polymer comprises 33.3%of said microparticle.
 19. The microparticle of claim 15, wherein said12.6 kDa polymer is an RG502H polymer.
 20. The microparticle of claim15, wherein said 55 kDa polymer is an RG505 polymer.
 21. Themicroparticle of claim 15, wherein said microparticle further comprisespolyethylene glycol.